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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide. To identify potential diagnostic markers for ESCC and therapeutic targets for ESCC, we used Serial Analysis of Gene Expression (SAGE) on one ESCC sample. We obtained a total of 14 430 tags, including 5765 that were unique. By comparing SAGE tags from the ESCC sample with those from normal human squamous esophagus, we found several genes that were differentially expressed between ESCC and normal squamous esophagus. Among these, we focused on the ADAM metallopeptidase with thrombospondin type 1 motif, 16 (ADAMTS16) gene because quantitative RT-PCR analysis showed a high level of ADAMTS16 expression in eight out of 20 ESCC samples (40%), but not in 15 kinds of normal tissues. Western blot analysis also showed upregulation of ADAMTS16 protein in ESCC tissues. Furthermore, ADAMTS16 protein was detected in culture media from the TE5 esophageal cancer cell line. Knockdown of ADAMTS16 in TE5 cells inhibited both cell growth and invasion ability. Our present SAGE data provide a list of genes potentially associated with ESCC. ADAMTS16 could be a novel diagnostic and therapeutic target for ESCC.

(Cancer Sci 2010; 101: 1038–1044)

Human esophageal cancer occurs worldwide with a variable geographic distribution and ranks eighth in order of occurrence and sixth as a leading cause of cancer mortality, affecting men more than women.(1) There are two main forms, each with distinct etiologic and pathologic characteristics, esophageal squamous cell carcinoma (ESCC) and adenocarcinoma. ESCC is the most frequent subtype of esophageal cancer, although the incidence of adenocarcinoma in the USA and UK is increasing faster than other esophageal malignancies. Most ESCC is diagnosed at an advanced stage, and even superficial ESCC that appears to extend no further than the submucosa metastasizes to the lymph nodes in 50% of cases.(2) In spite of the use of modern surgical techniques combined with various treatment modalities, such as chemoradiotherapy (CRT), the overall 5-year survival rate of ESCC still remains at 40–60%.(3) Therefore, identification of new diagnostic markers for ESCC and new therapeutic targets for ESCC is important.

Better knowledge of changes in gene expression that occur during carcinogenesis might lead to improvements in diagnosis, treatment, and prevention of ESCC. Genes encoding transmembrane/secretory proteins expressed specifically in cancers may be ideal diagnostic biomarkers.(4) Moreover, if the gene product functions in the neoplastic process, the gene is not just a biomarker but might also be a therapeutic target.(5) To identify potential markers for early detection of ESCC and therapeutic targets for ESCC, comprehensive gene expression analysis could be useful. Studies on differential global gene expression profiling in ESCCs using cDNA and oligonucleotide arrays have been carried out in various populations.(6,7) Although many studies have been done on gene expression profiling of specific tumor types, and differentially expressed genes in these tumors have been reported, few of these studies have resulted in clinical applications. However, among the comprehensive methods used to analyze transcript expression levels, Serial Analysis of Gene Expression (SAGE) is a common approach.(8) We previously carried out SAGE on four primary gastric cancer tissues(9) and identified several gastric cancer-specific genes.(10) Of these genes, regenerating islet-derived family, member 4 (REG4, which encodes Reg IV) and olfactomedin 4 (OLFM4, also known as GW112 or hGC-1) are highly sensitive serum markers for gastric cancer.(11,12) However, SAGE analysis on ESCC tissue has been done in only one case.(13)

In the present study, we generated the SAGE library from one ESCC sample. By comparing SAGE tags from ESCC samples with those from normal human squamous esophagus (Gene Expression Omnibus accession number, GSM52501),(14) we found several genes and tags that were differentially expressed between ESCC and normal squamous esophagus. Among these, we focused on the ADAM metallopeptidase with thrombospondin type 1 motif, 16 (ADAMTS16) gene because it is frequently overexpressed in ESCC, and ADAMTS16 expression is narrowly restricted among various normal tissues. In addition, the amino acid sequence of the ADAMTS16 protein suggests that it might be secreted. ADAMTS has been described as part of a family of zinc-dependent proteases (metzincin family) that play important roles in a variety of normal and pathological conditions, including arthritis and cancer.(15,16) Although expression of ADAMTS16 in some organs has been reported, the relationship with cancers, including ESCC, has not been studied.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Tissue samples.  For SAGE analysis, one primary ESCC (75-year-old male, T2N0M0) sample was used (Fig. 1). We confirmed microscopically that the tumor specimens consisted mainly (>80%) of carcinoma tissue. For quantitative RT-PCR analysis, 20 ESCC tissue samples and corresponding non-neoplastic mucosa samples were used. For Western blot analysis, four ESCC tissue samples and corresponding non-neoplastic mucosa samples were used. The samples were obtained from surgeries at Hiroshima University Hospital and affiliated hospitals. Samples were frozen immediately in liquid nitrogen and stored at −80°C until use. Fifteen kinds of normal tissue samples, including heart, lung, esophagus, stomach, small intestine, colon, liver, pancreas, kidney, bone marrow, peripheral leukocytes, spleen, skeletal muscle, brain, and spinal cord, were purchased from Clontech (Palo Alto, CA, USA). Histological classification was based on the World Health Organization system. Tumor staging was done according to the TNM stage grouping system.(17) For strict privacy protection, identifying information for all samples was removed before analysis. This procedure was in accordance with the Ethical Guidelines for Human Genome/Gene Research of the Japanese Government.

image

Figure 1.  Histological features of the esophageal squamous cell carcinoma sample analyzed by Serial Analysis of Gene Expression. The formalin-fixed, paraffin-embedded section was stained with H&E.

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Serial analysis of gene expression.  SAGE was carried out according to SAGE protocol version 1.0e (June 23, 2000). Tags were extracted from the raw sequence data with SAGE2000 analysis software version 4.12, kindly provided by Dr. Kenneth W. Kinzler (Ludwig Center for Cancer Genetics and Therapeutics and Howard Hughes Medical Institute, Johns Hopkins Kimmel Cancer Center, Baltimore, MD, USA).

Quantitative RT-PCR.  Total RNA was extracted with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and 1 μg total RNA was converted to cDNA with a First Strand cDNA Synthesis Kit (Amersham Biosciences, Piscataway, NJ, USA). PCR was carried out with a SYBR Green PCR Core Reagents Kit (Applied Biosystems, Foster City, CA, USA). ADAMTS16 primer sequences were 5′-TCT CAT AGG AGT CGC CTC TGC-3′ and 5′-CGA GTG GAG CCC TCA CAG AA-3′. Squamous cell carcinoma antigen A1 (SCCA1) primer sequences were 5′-GAA TGG TGG ATA TCT TCA ATG GG-3′ and 5′-GAT AGC ACG AGA CCG CGG-3′. Real-time detection of the emission intensity of SYBR Green bound to double-stranded DNA was done with an ABI PRISM 7700 Sequence Detection System (Applied Biosystems) as described previously.(18) Actin-beta-specific PCR products were amplified from the same RNA samples and served as internal controls.

Cell line and RNAi.  Human esophageal cancer-derived cell lines, TE1, TE3, TE5, TE7, and TE13, were kindly provided by Dr. Tetsuro Nishihara (Tohoku University School of Medicine, Miyagi, Japan).(19) All cell lines were maintained in RPMI-1640 (Nissui Pharmaceutical, Tokyo, Japan) containing 10% FBS (Whittaker, Walkersville, MD, USA) in a humidified atmosphere of 5% CO2 and 95% air at 37°C. To knockdown the endogenous ADAMTS16, RNAi was carried out. siRNA oligonucleotides for ADAMTS16 and a negative control were purchased from Invitrogen (Carlsbad, CA, USA). Three independent oligonucleotides were used for ADAMTS16 siRNA. The ADAMTS16 siRNA1 sequence was 5′-CCA GUA UUA UCA CAU GGU CAC CAU U-3′. The ADAMTS16 siRNA2 sequence was 5′-ACA GAG ACC UGA AGU UUC AAG UAA A-3′. The ADAMTS16 siRNA3 sequence was 5′-GAG UAU AAG UCU UGC UUA CGG CAU A-3′. Transfection was carried out using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. Briefly, 60 pmol siRNA and 10 μL Lipofectamine RNAiMAX were mixed in 1 mL RPMI medium (10 nmol/L final siRNA concentration). After 20 min of incubation, the mixture was added to the cells and these were plated on dishes for each assay. Forty-eight hours after transfection, cells were analyzed for all experiments.

Western blot analysis.  For Western blot analysis, tissue samples or cells were lysed as described previously.(20) The culture media were concentrated with the Protein Concentrate Kit (Takara Bio, Shiga, Japan). The lysates (40 μg) were solubilized in Laemmli sample buffer by boiling, then subjected to 8% SDS-PAGE followed by electrotransfer onto a nitrocellulose filter. The filter was incubated with the primary antibody against ADAMTS16 (rabbit polyclonal, dilution 1:500; Abcam, Cambridge, UK). Peroxidase-conjugated antirabbit IgG was used in the secondary reaction. Immunocomplexes were visualized with an ECL Western Blot Detection System (Amersham Biosciences). β-actin antibody (Sigma Chemical, St. Louis, MO, USA) was also used as a loading control.

Cell growth and in vitro invasion assays.  The cells were seeded at a density of 2000 cells per well in 96-well plates. Cell growth was monitored after 1 and 2 days by MTT assay.(21) Modified Boyden chamber assays were carried out to examine invasiveness. Cells were plated at 10 000 cells per well in RPMI-1640 medium plus 1% serum in the upper chamber of a Transwell insert (8 μm pore diameter; Chemicon, Temecula, CA, USA) coated with Matrigel. Medium containing 10% serum was added in the bottom chamber. After 1 and 2 days, cells in the upper chamber were removed by scraping, and the cells remaining on the lower surface of the insert were stained with CyQuant GR dye (Chemicon, Temecula, CA, USA) to assess the number of cells.

Statistical methods.  Correlations between clinicopathologic parameters and ADAMTS16 mRNA expression were analyzed by Fisher’s exact test. A P value of <0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Generation of SAGE data and comparison of expression patterns in ESCC and normal squamous esophagus.  A total of 14 430 tags was generated, including 5765 that were unique. Then we compared SAGE tags from the ESCC sample with those from normal squamous esophagus (Gene Expression Omnibus accession number, GSM52501), which contained a total of 50 508 tags including 14 835 unique tags. The 20 most upregulated tags and the 20 most downregulated tags are shown in Tables 1 and 2. The upregulated tags included ADAMTS16, immunoglobulin heavy constant gamma 1 (IGHG1), 2-oxoglutarate and iron-dependent oxygenase domain containing 1 (OGFOD1), nuclear transport factor 2 (NUTF2), and RING1 and YY1 binding protein (RYBP), whose expressions have not been investigated in ESCC. The downregulated tags included S100 calcium binding protein A9 (S100A9), keratin 4 (KRT4), cystatin B (CSTB), exportin 7 (XPO7), keratin 6C (KRT6C), and epithelial membrane protein 1 (EMP1). Downregulation of some of these genes has been reported previously.(13) To identify novel biomarkers for ESCC diagnosis and novel targets for ESCC treatment, we focused on genes that were upregulated in the ESCC sample. Of the upregulated genes, we decided to analyze ADAMTS16 expression because the amino acid sequence of the ADAMTS16 protein suggests that it might be secreted.

Table 1.   Twenty most upregulated tags in esophageal squamous cell carcinoma (ESCC) compared to normal squamous esophagus (normal)
Tag sequenceTags per millionSymbolDescription
ESCCNormal
  1. †Absolute tag counts are normalized to 1 000 000 total tags/sample. ‡Number in parentheses indicates the absolute tag counts.

TCCCCTACAT2564† (37)‡0 (0)ADAMTS16ADAM metallopeptidase with thrombospondin type 1 motif, 16
GAAATAAAGC2495 (36)0 (0)IGHG1Immunoglobulin heavy constant gamma 1 (G1m marker)
TTCGGTTGGT2148 (31)0 (0)OGFOD12-Oxoglutarate and iron-dependent oxygenase domain containing 1
AGGCATTGAA5336 (77)20 (1)NUTF2Nuclear transport factor 2
CAGTTACAAA5544 (80)40 (2)RYBPRING1 and YY1 binding protein
TGGAAATGAC1317 (19)0 (0)COL1A1Collagen, type I, alpha 1
GGCGTTTAGA2079 (30)20 (1)No matchNo match
ACCAAAAACC1663 (24)20 (1)COL1A1Collagen, type I, α1
GGCAGCACAA1455 (21)20 (1)NBEAL2Neurobeachin-like 2
TTTATTAGAA1455 (21)20 (1)CCDC75Coiled-coil domain containing 75
AGCCAAAAAA2980 (43)40 (2)MAP3K12Mitogen-activated protein kinase kinase kinase 12
GCTTTCATTG2495 (36)40 (2)NUCKSNuclear casein kinase and cyclin-dependent kinase substrate 1
   GPX2Glutathione peroxidase 2 (gastrointestinal)
ATGTGAAGAG901 (13)0 (0)SPARCSecreted protein, acidic, cysteine-rich (osteonectin)
CTCCCCCAAA693 (10)0 (0)KLK10Kallikrein-related peptidase 10
   IGHA2Immunoglobulin heavy constant α2 (A2m marker)
GCTTAAAAAA693 (10)0 (0)CORO1CCoronin, actin binding protein, 1C
ATTTGAGAGT624 (9)0 (0)MYH9Myosin, heavy chain 9, non-muscle
CTTTATTCCA624 (9)0 (0)WWC2WW and C2 domain containing 2
TCAAGCCATC624 (9)0 (0)BLMHBleomycin hydrolase
   PCYT2Phosphate cytidylyltransferase 2, ethanolamine
TTTTCCAATT624 (9)0 (0)UTP3UTP3, small subunit (SSU) processome component, homolog (S. cerevisiae)
TTGCTCACAA1178 (17)20 (1)ABHD12BAbhydrolase domain containing 12B
Table 2.   Twenty most downregulated tags in esophageal squamous cell carcinoma (ESCC) compared to normal squamous esophagus (normal)
Tag sequenceTags per millionSymbolDescription
ESCCNormal
  1. †The absolute tag counts are normalized to 1 000 000 total tags/sample. ‡Number in parentheses indicates the absolute tag counts.

GTGGCCACGG0 (0)25 283† (1277)‡S100A9S100 calcium binding protein A9 (calgranulin B)
GGCAGAGAAG0 (0)8454 (427)KRT4Keratin 4
ATGAGCTGAC0 (0)3762 (190)CSTBCystatin B (stefin B)
XPO7Exportin 7
GAAGCACAAG0 (0)2475 (125)KRT6CKeratin 6C
TAATTTGCAT0 (0)2455 (124)EMP1Epithelial membrane protein 1
GNA13Guanine nucleotide binding protein (G protein), α 13
AAAGCGGGGC0 (0)2356 (119)KRT13Keratin 13
TGTGTTGAGA0 (0)2257 (114)EEF1A1Eukaryotic translation elongation factor 1 α 1
CACAAACGGT0 (0)2079 (105)TSPAN9Tetraspanin 9
RPS27Ribosomal protein S27
TGGTGTTGAG0 (0)1841 (93)RPS18Ribosomal protein S18
GCCAATCCAG0 (0)1802 (91)CRNNCornulin
GGCAAGCCCC0 (0)1782 (90)RPL10ARibosomal protein L10a
PTPRGProtein tyrosine phosphatase, receptor type, G
AAGGAGATGG0 (0)1722 (87)RPL31Ribosomal protein L31
ZNF434Zinc finger protein 434
CTGTCACCCT0 (0)1564 (79)SPRR1ASmall proline-rich protein 1A
BTCBetacellulin
TAAGGAGCTG0 (0)1485 (75)RPS26Ribosomal protein S26
ANK2Ankyrin 2, neuronal
ACCTGGAGGG0 (0)1386 (70)SBSNSuprabasin
PCBP1Poly(rC) binding protein 1
ACGTGTGTAA0 (0)1386 (70)No matchNo match
CAAATCCAAA0 (0)1366 (69)No matchNo match
GCCGAGGAAG0 (0)1346 (68)RPS12Ribosomal protein S12
NCKAP5LNCK-associated protein 5-like
TGTGCTAAAT0 (0)1346 (68)USP36Ubiquitin specific peptidase 36
RPL34Ribosomal protein L34
GGGTCTGAGG0 (0)1307 (66)SLURP1Secreted LY6/PLAUR domain containing 1
PTPRGProtein tyrosine phosphatase, receptor type, G

mRNA expression of ADAMTS16.  Because genes expressed at high levels in tumors and at greatly reduced levels in normal tissues are ideal diagnostic markers and therapeutic targets,(4) quantitative RT-PCR of ADAMTS16 was carried out in 20 ESCC samples and in 15 kinds of normal tissue (liver, kidney, heart, colon, brain, bone marrow, skeletal muscle, lung, small intestine, spleen, spinal cord, stomach, pancreas, leukocyte, and esophagus) (Fig. 2a). Among the various normal tissues, obvious ADAMTS16 expression was found in normal brain, spinal cord, pancreas, and kidney, as reported elsewhere.(22) Expression of ADAMTS16 in these normal tissues was highest in spinal cord; however, in ESCC, high levels of ADAMTS16 mRNA expression (more than twice the mRNA expression of spinal cord) were found in eight out of 20 cases (40%). ADAMTS16 expression in two ESCC cases (Cases 16 and 17) was 10-fold higher than in spinal cord. High levels of ADAMTS16 mRNA expression were not correlated with any clinicopathologic characters (data not shown). Among five cases at stage I ESCC, a high level of ADAMTS16 mRNA was detected in one case (20%). These results indicate that ADAMTS16 expression is highly specific for cancer, at least in ESCC.

image

Figure 2.  Quantitative RT-PCR analysis of ADAMTS16 and SCCA1 in 15 kinds of normal tissues and 20 esophageal squamous cell carcinoma tissues. (a) mRNA expression level of ADAMTS16. The units are arbitrary, and we calculated ADAMTS16 mRNA expression by standardization of the expression in normal spinal cord to 1.0. (b) mRNA expression level of SCCA1. The units are arbitrary, and we calculated SCCA1 mRNA expression by standardization of the expression in normal lung to 1.0.

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Serum squamous cell carcinoma antigen (SCC antigen) detected in the normal squamous epithelium and in ESCC has been considered a useful tumor marker for ESCC.(23) SCC antigen predicts recurrence or progression of the disease and has been used extensively for this purpose. However, clinical use of this marker has been restricted because of lack of sensitivity.(24) Therefore, there is an urgent need for new biomarkers for ESCC. To evaluate the usefulness of determining ADAMTS16 expression as a tumor marker, we measured expression levels of SCC antigen and compared them with ADAMTS16 levels. Because a measurement system for serum levels of ADAMTS16 is not available, we investigated the mRNA expression levels of SCCA1, which encodes SCC antigen, by quantitative RT-PCR (Fig. 2b). In 15 kinds of normal tissue, expression of SCCA1 was highest in lung; however, in ESCC, high levels of SCCA1 mRNA expression (more than twice the mRNA expression levels of lung) were found in four of 20 cases (20%). Among five cases at stage I ESCC, high levels of SCCA1 mRNA were not detected. These results indicate that ADAMTS16 might serve as a more sensitive biomarker than SCC antigen. We calculated the ratio of ADAMTS16 mRNA expression levels between ESCC tissue (T) and corresponding non-neoplastic mucosa (N). T/N ratios >2-fold higher were considered to represent overexpression. ADAMTS16 overexpression was observed in 13 of 20 ESCC cases (65%). Among five cases at stage I ESCC, ADAMTS16 overexpression was found in one case (20%). We then investigated the relation of ADAMTS16 expression to clinicopathologic characters (Table 3). We found that ADAMTS16 overexpression correlated to the advanced T classification and tumor stage.

Table 3.   Relationship between ADAMTS16 expression and clinico-pathologic characteristics in esophageal squamous cell carcinoma
 ADAMTS16 expressionP value*
OverexpressionNo overexpression
  1. *Fisher’s exact test. N, node; T, tumor.

Age (years)
 ≤658 (80%)2 (20%)0.3498
 >655 (50%)5 (50%)
Sex
 Male11 (69%)5 (31%)0.5868
 Female2 (50%)2 (50%)
T classification
 T12 (29%)5 (71%)0.0215
 T2/311 (85%)2 (15%)
N classification
 N03 (43%)4 (57%)0.1736
 N110 (77%)3 (23%)
Stage
 Stage I1 (20%)4 (80%)0.0307
 Stage II/III12 (80%)3 (20%)

ADAMTS16 protein expression.  Analysis of the amino acid sequence of the ADAMTS16 protein suggests that it might be secreted. To investigate whether ADAMTS16 is a secreted protein, we used Western blot analysis in five esophageal cancer cell lines. Moderate to high ADAMTS16 expression was noted in TE1, TE3, and TE5 cells as a band of approximately 136 kDa, and the other two remaining cell lines (TE7 and TE13) had low or absent ADAMTS16 expression (Fig. 3a). Next, we examined the transition of ADAMTS16 expression by Western blot analysis of cell extracts of TE5 transfected with ADAMTS16 specific siRNAs. Three types of siRNAs (siRNA1–3) were transfected into TE5. The expression of ADAMTS16 in TE5 was substantially suppressed by treatment with siRNA2 and siRNA3, but not with siRNA1 (Fig. 3b). Therefore, to knock down the endogenous ADAMTS16, we used siRNA2 and siRNA3 in the following experiments. A Western blot was carried out of siRNA (siRNA2 and siRNA3)-transfected TE5 cell extracts and culture media (Fig. 3c). In negative control siRNA-transfected TE5 cells, ADAMTS16 protein was detected in culture media as well as cell extracts; however, in ADAMTS16 siRNA-transfected TE5 cells, ADAMTS16 protein was low or absent in culture media as well as cell extracts. These results clearly indicate that ADAMTS16 is a secreted protein.

image

Figure 3.  ADAMTS16 protein expression and functional analysis. (a) Western blot analysis of ADAMTS16 in five esophageal squamous cell carcinoma (ESCC) cell lines. (b) Western blot analysis of ADAMTS16 in cell lysates from TE5 cells transfected with the negative control siRNA and ADAMTS16 siRNA (siRNA1–3). (c) Western blot analysis of ADAMTS16 in cell lysates and culture media from TE5 cells transfected with the negative control siRNA and ADAMTS16 siRNA (siRNA2 and 3). (d) Western blot analysis of ADAMTS16 in four ESCC samples (T) and corresponding non-neoplastic mucosa samples (N). (e) Effect of ADAMTS16 knockdown on cell growth of TE5 and TE7 cells. Cell growth was assessed by an MTT assay at 1, 2, 4, and 8 days after seeding on 96-well plates. Bars and error bars, mean and SE of three different experiments. O.D., optical density. (f) Effect of ADAMTS16 knockdown on cell invasion of TE5 and TE7 cells. TE5 and TE7 cells transfected with negative control siRNA and ADAMTS16 siRNA (siRNA2 and 3) were incubated in Boyden chambers. After 1 and 2 days, invading cells were counted. Bars and error bars, mean and SE of three different experiments. N.S., not significant. *P = 0.0006; **P = 0.0003; ***P < 0.0001.

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Next, expression of ADAMTS16 protein was analyzed by a Western blot of four ESCC tissue samples and corresponding non-neoplastic mucosa samples (Fig. 3d). Among the four ESCC samples, ADAMTS16 protein expression was detected in all; however, of the four corresponding non-neoplastic mucosa samples, ADAMTS16 protein expression was found in only one sample. These results indicate that ADAMTS16 protein is overexpressed in ESCC tissue, and can serve as a serum tumor marker for ESCC.

Effect of ADAMTS16 inhibition on cell growth and invasive activity of esophageal cancer cells.  High levels of ADAMTS16 mRNA expression were correlated with T classification of ESCC tissues; however, the biological significance of ADAMTS16 in ESCC has not been studied. To investigate the possible antiproliferative effects of ADAMTS16 knockdown, we carried out an MTT assay 8 days after siRNA transfection (Fig. 3e). TE5 cells were selected for high ADAMTS16 expression. ADAMTS16 siRNA2-transfected and siRNA3-transfected TE5 cells showed significantly reduced viability relative to negative control siRNA-transfected TE5 cells. We carried out the same assay using one additional esophageal cancer cell line that did not express ADAMTS16 (TE7). Reduced cell viability was not observed in siRNA2- or siRNA3-transfected TE7 cells compared with negative control siRNA-transfected TE7 cells.

Next, to determine the possible role of ADAMTS16 in the invasiveness of esophageal cancer cells, we used a Transwell invasion assay (Fig. 3f). On day 1, although there was no difference in cell viability between ADAMTS16 knockdown TE5 cells and negative control siRNA-transfected TE5 cells, the invasiveness of ADAMTS16 knockdown TE5 cells was 40% less than that of the negative control siRNA-transfected TE5 cells. On day 2, the invasiveness of ADAMTS16 knockdown TE5 cells was 50% less than that of the negative control siRNA-transfected TE5 cells; however, as ADAMTS16 knockdown cells showed significantly reduced cell viability, the cell number difference observed in the invasion assay might be caused by the reduced cell viability. In contrast, invasion ability was not significantly different between ADAMTS16 knockdown TE7 cells and negative control siRNA-transfected TE7 cells. These results indicate that ADAMTS16 stimulates cell growth and invasion in esophageal cancer cells.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

In spite of improvement to modern surgical techniques and adjuvant CRT, ESCC is known to reveal the worst prognosis among malignant tumors. Therefore, it is now urgently required to develop novel diagnostic biomarkers and therapeutic targets for a better choice of adjuvant treatment modalities for individual patients. In the present study, we carried out a genome-wide expression profile analysis of one ESCC tissue sample by SAGE, and identified upregulated and downregulated genes in ESCC. Among these, we further investigated ADAMTS16. Quantitative RT-PCR revealed that ADAMTS16 mRNA expression was frequently upregulated in ESCC, and was narrowly restricted in normal tissues. Western blot analysis also showed upregulation of ADAMTS16 protein in ESCC. Furthermore, ADAMTS16 protein was detected in culture media from TE5 cells. Taken together, these results suggest that ADAMTS16 has potential as a serum tumor marker for ESCC. Because the frequency of high levels of ADAMTS16 mRNA expression (40%) was greater than the frequency of high levels of SCCA1 mRNA expression (20%), serum concentrations of ADAMTS16 might serve as a sensitive biomarker for ESCC. In contrast, because ADAMTS16 mRNA overexpression was correlated with advanced T classification and tumor stage, serum concentrations of ADAMTS16 might not be suitable for early detection of ESCC. Serum concentrations of ADAMTS16 should be measured in patients with ESCC.

In the present study, ADAMTS16 mRNA overexpression correlated to the advanced T classification and tumor stage. Knockdown of ADAMTS16 by RNAi inhibited the cell growth and invasion ability of TE5 cells. Because expression of ADAMTS16 was highly specific to ESCC, it could be a good therapeutic target with less adverse effects for ESCC. Although the function of ADAMTS16 is poorly understood, members of the metzincin family are known to process a number of growth factors, cytokines and signaling molecules in addition to matrix substrates.(25) However, it has been reported that the forced expression of ADAMTS16 has no effect on expression levels of most of the ADAMTS, TIMP, and MMP genes. In the present study, we also used ELISA to measure levels of epidermal growth factor (EGF) and transforming growth factor (TGF)-α in culture media from TE5 cells transfected with ADAMTS16 siRNA and negative control siRNA; however, levels of EGF and TGF-α were not significantly different (data not shown). Therefore, growth factors or cytokines, such as EGF or TGF-α, are not likely to be involved in mechanisms of cell growth inhibition and invasion ability following knockdown of ADAMTS16.

Although ADAMTS16 protein upregulation was observed in ESCC tissues by Western blot analysis, expression and distribution of ADAMTS16 protein in ESCC tissues remains unclear. Therefore, immunohistochemical analysis should be undertaken. Unfortunately, the antibody against ADAMTS16 used in the present study is not suitable for immunostaining because the antibody against ADAMTS16 detected multiple bands on Western blots. Production of a specific antibody against ADAMTS16 is required. Furthermore, ADAMTS16 expression at mRNA and protein levels should be examined in several more tissues from stage I ESCC in the near future.

In addition to ADAMTS16, other upregulated and downregulated genes in ESCC were found. The upregulated group of genes identified by SAGE contains genes whose expression has not been investigated in ESCC. Upregulation of two genes related to the immunoglobulin heavy chain (IGHG1 and IGHA2) was found in the present study. Previously, genes involved in the immune response have been shown as characteristically upregulated in long-term ESCC survivors who were treated with CRT.(26) Therefore, the ESCC case analyzed by SAGE in the present study might be sensitive to CRT. OGFOD1 is a 2-oxoglutarate and Fe(II)-dependent oxygenase, a class of enzymes that catalyze a variety of reactions typically involving the oxidation of an organic substrate using a dioxygen molecule.(27) To our knowledge, association between cancer and OGFOD1 has not been investigated. NUTF2 encodes nuclear transport factor 2 (NTF2), which is a small GDP Ran binding protein. The main function of NTF2 is to facilitate transport of certain proteins into the nucleus through interaction with nucleoporin FxFG.(28) It is also involved in regulating multiple processes, including cell cycle and apoptosis.(29) However, no studies have analyzed NTF2 expression in human cancer, including ESCC. RYBP is a member of the polycomb group, and it has been reported that RYBP interacts with MDM2 and decreases MDM2-mediated p53 ubiquitination, leading to stabilization of p53 and an increase in p53 activity.(30) RYBP induces cell cycle arrest and is involved in the p53 response to DNA damage. Expression of RYBP is decreased in hepatocellular carcinoma and lung cancer tissues.(30) Therefore, upregulation of RYBP should be confirmed in a large number of ESCC cases. In contrast, downregulated genes identified by SAGE in the present study were similar to genes previously reported as downregulated in ESCC.(13)

In conclusion, our present SAGE data provide a list of genes potentially associated with ESCC. Because our list is based on one ESCC case, expression analysis in a large number of cases is required. A high level of ADAMTS16 expression was detected in ESCC, and expression of ADAMTS16 was narrowly restricted. Production of a specific antibody against ADAMTS16 protein and establishment of a measurement system for serum samples are needed to clarify whether ADAMTS16 serves as a serum marker for early detection and a good therapeutic target for ESCC.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

We thank Mr. Shinichi Norimura for excellent technical assistance and advice. We thank the Analysis Center of Life Science, Hiroshima University (Hiroshima, Japan) for the use of their facilities. This work was supported, in part, by Grants-in-Aid for Cancer Research from the Ministry of Education, Culture, Science, Sports, and Technology of Japan, partly by a Grant-in-Aid for the Third Comprehensive 10-Year Strategy for Cancer Control and for Cancer Research from the Ministry of Health, Labour, and Welfare of Japan, and partly by a grant (07-23911) from the Princess Takamatsu Cancer Research Fund.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References