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Frequent Methylation-Associated Silencing of the Tissue Inhibitor of Metalloproteinase-3 Gene in Pancreatic Endocrine Tumors

Departments of Surgery (A.W., P.L., V.F., B.C., D.K.B.) and Pathology (A.R.), Institute of Theoretical Surgery (I.C.), and Department of Internal Medicine (B.S.), Philipps-University Marburg, 35043 Marburg, Germany

Address all correspondence and requests for reprints to: Detlef K. Bartsch, M.D., Department of Surgery, Philipps-University Marburg, Baldingerstrasse, D-35043 Marburg, Germany. E-mail: bartsch{at}mailer.uni-marburg.de.

	Abstract

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Molecular mechanisms contributing to the tumorigenesis of pancreatic endocrine tumors (PETs) are still not well understood. Allelic deletions at chromosome 22q12.3 were detected in about 30–60% of PETs, suggesting that inactivation of one or more tumor suppressor genes on this chromosomal arm is important for their pathogenesis. Because the putative tumor suppressor gene tissue inhibitor of metalloproteinase-3 (TIMP-3) has been located at 22q12.3, we undertook a genetic analysis of TIMP-3 to determine its role in the tumorigenesis of PETs. Single-strand conformational polymorphism analysis, methylation-specific PCR, RNA expression analysis, and immunohistochemistry of TIMP-3 were performed in 21 sporadic PETs. Thirteen of 21 PETs (62%) revealed TIMP-3 alterations, including promoter hypermethylation and homozygous deletion. The predominant TIMP-3 alteration was promoter hypermethylation, identified in 8 of 18 (44%) PETs. It was tumor-specific and corresponded to loss or strong reduction of TIMP-3 protein expression. Notably, 11 of 14 (79%) PETs with metastases had TIMP-3 alterations, compared with only 1 of 7 (14%) PETs without metastases (P < 0.02). These data suggest a possibly important role of TIMP-3 in the tumorigenesis of human PETs, especially in the development of metastases, which has to be further evaluated in large-scale studies.

	Introduction

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PANCREATIC ENDOCRINE TUMORS (PETs) represent a distinct group of rare tumors that are frequently associated with unique clinical syndromes that result from an excess of endogenously produced hormones, such as insulin or gastrin (1). Due to the rare occurrence of PETs, our knowledge of genetic changes associated with the initiation and progression of these neoplasms is still limited, although significant knowledge has been gained in the last 5 yr. Important genetic findings identified in human PETs are summarized in Table 1 (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). In particular, we and others detected significant loss of heterozygosity (LOH) at chromosome 22q12.3 in about 30% of insulinomas (19) and 35–59% of gastrinomas and nonfunctioning neuroendocrine carcinomas (17, 18, 20). These data indicated the possible presence of one or more tumor suppressor genes at this chromosomal region that might be involved in the tumorigenesis of PETs. One putative tumor suppressor gene at chromosome 22q12.3 is the tissue inhibitor of metalloproteinase-3 (TIMP-3) gene. TIMPs are a family of secreted molecules that inhibit the proteolytic activity of the matrix metalloproteinases (MMPs; Ref. 21). Tumors frequently show an increase in MMP expression and/or decrease of TIMP expression leading to an imbalance in proteolytic activity during tumor progression. TIMP-3 is a secreted 24-kDa protein that, unlike other TIMP family members, binds to the extracellular matrix. TIMP-3 antagonizes the activity of MMPs by binding covalently to the active site of the enzymes (22). It is thought that reduced expression of TIMP-3 contributes to primary tumor growth, angiogenesis, apoptosis, tumor invasion, and metastasis by allowing increased activity of MMPs in the extracellular matrix (23, 24, 25). Recent studies on methylation-associated silencing of TIMP-3 suggested a tumor suppressor role in kidney, brain, breast, and colon cancers (26). In the present study, the involvement of TIMP-3 in the tumorigenesis of 21 human PETs was evaluated. We demonstrate for the first time frequent alterations of TIMP-3 in these neoplasms, primarily targeted by tumor-specific methylation-associated silencing leading to lack of protein expression.

	Materials and Methods

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Twenty-one fresh-frozen sporadic PETs were obtained from the tumor bank of the Department of Surgery, Philipps-University Marburg, including nine nonfunctioning neuroendocrine pancreatic carcinomas (NNPCs), five insulinomas, five gastrinomas, one vipoma, and one renin-producing PET (27). The diagnosis of insulinoma required a symptomatic hypoglycemia (<40 mg/dl) with concomitant endogenous hyperinsulinemia (>0.12 nmol/liter) during a supervised fast and a positive immunohistochemistry of the tumor for insulin. The diagnosis of Zollinger-Ellison syndrome was established by clinical symptoms, an elevated fasting serum gastrin level (>125 pg/ml) in the presence of acid in the stomach, a positive secretin stimulation test defined as increase to a serum gastrin concentration of more than 200 pg/ml, and a positive immunohistochemistry for gastrin in the tumor. The diagnosis of vipoma was confirmed by secretory diarrhea (>6 liter/d) and a fasting vasoactive intestinal polypeptide concentration greater than 130 pg/ml. Tumors were considered as nonfunctioning if clinically no symptoms of hormonal excess were present and plasma hormone levels were within normal limits. Malignancy was determined on the basis of the strict criteria of infiltrating overgrowth, lymph node, or distant metastases. Clinical follow-up was obtained through the patient’s personal physician or at outpatient attendance. Survival was calculated from the time of surgical resection to either death or most recent contact. Informed consent was obtained from all patients. All investigations and all patient material in this study were assessed under a research protocol approved by the Philipps-University of Marburg Ethic Committee. Tumor samples used for DNA and RNA isolation had a neoplastic cellularity between 85% and 100% after cryostat microdissection, whereas constitutional normal DNA was derived from peripheral blood lymphocytes or normal tissue specimens if available. Genomic DNA from fresh-frozen tissue and whole blood samples was isolated using the QIAamp DNA kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol, whereas total RNA was extracted with the TRIzol RNA kit as advised by the manufacturer (Life Technologies, Inc., Karlsruhe, Germany).

Single-strand conformational polymorphism (SSCP) and DNA sequence analysis of TIMP-3

The oligonucleotides for SSCP analysis of exons 1 to 5 of the TIMP-3 gene have been chosen from the published National Center for Biotechnology Information sequence (accession no. AH003680) as follows— Ex1For: 5'-gcacggcaactttggaga-3'; Ex1Rev: 5'-ggcaccaggagcgcttac-3'; Ex2For: 5'-ctgatgtggttcaggccttc-3'; Ex2Rev: 5'-cactttgcctcccttagcag-3'; Ex3For: 5'-acccagcagtggattaggac-3'; Ex3Rev: 5'-caaaccctggcctagaagc-3'; Ex4For: 5'-gctgcaggaattctctctgg-3'; Ex4Rev: 5'-ccagtgaccccagagcttac-3'; Ex5For: 5'-gagtccatcaactgctgcctg-3'; and Ex5Rev: 5'-gaagggagggaagtgaggtg-3'. PCR amplification and SSCP analysis were performed as described previously (19).

Methylation-specific PCR (MSP) assay

Genomic DNA was extracted as mentioned above, and 1 µg of DNA was bisulfite-modified as described by Herman et al. (28). The sequence difference of bisulfite-treated DNA was detected by PCR using primers specific for either the methylated or unmethylated DNA. Oligonucleotide sequences and PCR conditions were chosen according to Ueki et al. (29). MSP primer sequences that specifically recognized unmethylated TIMP-3 sequence were 5'-TTTTGTTTTGTTATTTTTTGTTTTTGGTTTT-3' (upstream) and 5'-CCCCCCAAAAACCCCACCTCA-3' (downstream); and the methylated TIMP-3 sequence 5'-CGTTTCGTTATTTTTTGTTTTCGGTTTTC-3' (upstream) and 5'-CCGAAAACCCCGCCTCG-3' (downstream). MSP was performed on 2 µl bisulfite-treated DNA under the following conditions: 96 C for 3 min; 40 cycles of 96 C for 30 sec, 59 C for 30 sec, and 72 C for 30 sec; and a final extension of 3 min at 72 C. DNA from peripheral blood lymphocytes treated with Sss I methylase (New England Biolabs, Inc., Beverly, MA) was used as a positive control for methylated alleles. Ten microliters of each PCR product were loaded onto 6% polyacrylamide mini gels, ethidium bromide stained, and visualized under UV transillumination.

Immunohistochemistry

Immunohistochemical stainings for TIMP-3 were performed by a standard avidin-biotinylated immunohistochemistry technique using a commercial rabbit antihuman TIMP-3 polyclonal antibody (Chemicon International, Inc., Temecula, CA) and following a standard protocol (26). Each experiment contained normal pancreas and normal kidney as positive controls. Staining results of tumor sections were interpreted by an experienced pathologist (A.R.) without knowledge of clinical and molecular data. The TIMP-3 results were scored as follows: -, negative; +, less than 10% of cells positive; ++, 10–50% of tumor cells positive; +++, more than 50% of tumor cells positive.

RT-PCR of TIMP-3

Enough material for total RNA preparation was available for only 12 PETs (4 insulinomas, 4 gastrinomas, 3 NNPCs, and 1 renin-producing PET). RT-PCR analysis was performed with the One-step RT-PCR kit as described by the manufacturer’s protocol (Life Technologies, Inc.) with some modifications (29). The oligonucleotides for TIMP-3 RT-PCR, coding for a 648-bp amplicon, were chosen as follows: 5'-GGAATTCATGACCCCTTGGCTCGGG-3' (upstream) and 5'-ggaattcagggtctggcgctcaggg-3' (downstream). Integrity of RNA was verified by ß-actin amplification with the following oligonucleotides: (upstream) 5'-GATGATGATATCGCCGCGCTCGTCGTC-3' and (downstream) 5'-GTGCCTCAGGGCAGCGGAACCGCTCA-3' (778-bp fragment; Ref. 30). The products were visualized on 6% polyacrylamide mini gels, followed by ethidium bromide staining.

LOH analysis on 22q12.3

The LOH data on chromosome 22q12.3 has been published for most of the tumors previously (20). In brief, microsatellite markers D22S280 and D22S283 have been chosen for determination of LOH at chromosome 22q12.3. PCR amplification was performed with fluorescence-labeled oligonucleotides on a MWG Primus 25 PCR cycler (MWG Biotech, Ebersberg, Germany) with a standard protocol. Semiquantitative PCR product analysis was performed on a 310 Genetic Analyzer (PE Applied Biosystems, Foster City CA) as described by the manufacturer. Only microsatellites showing two distinct alleles in normal DNA were considered as informative. LOH was defined as a reduction in intensity of one allele in the tumor sample with respect to the matched allele from corresponding normal tissue. LOH was scored when the relative intensities of the tumor alleles compared with the corresponding normal tissue alleles differed by at least a factor of two, symbolizing a reduction of at least 50% or more in either of the two tumor alleles. Data were analyzed using Genescan version 2.1.1 software (PE Applied Biosystems).

Statistics

Descriptive statistics were expressed as mean and SD value system. The Fisher’s exact test was performed for nominal data. P values less than 0.05 were considered as statistically significant. Data were analyzed using SPSS software (version 10; SPSS, Inc., Chicago, IL).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
We screened the putative tumor suppressor gene TIMP-3 for alterations in 21 sporadic PETs, including 9 NNPCs, 5 insulinomas, 5 gastrinomas, 1 vipoma, and 1 renin-producing PET (27). Thirteen males and 8 females with a median age of 52 (range, 28–66) yr at the time of surgery were included in this study. The tumor size ranged from 5 mm to 350 mm in diameter. Sixteen tumors were malignant, whereas three insulinomas and two gastrinomas appeared to be benign. At the time of surgery, 10 patients had localized disease, defined by the absence of metastases. Eight patients had tumors with lymph node metastases, and three patients presented with synchronous liver metastasis. During median follow-up of 69 (range, 2–339) months, five patients developed liver metastases. At conclusion of the study, 12 patients had no evidence of disease, 4 patients were alive with disease, and 5 patients were deceased due to diffuse liver metastases.

The TIMP-3 gene was screened for mutations by SSCP analysis in all 21 PETs. Exons 1 to 5 were successfully amplified in all tumors but NNPC 166/98. This was suggestive for a homozygous deletion of TIMP-3 in this sample, because the DNA integrity was confirmed in several control experiments, including the successful amplification of K-ras, p53, and p16^INK4 genes (data not shown). SSCP analysis revealed no variant bands in any of the 21 PETs. Thus, no sequence alterations in the coding regions of TIMP-3 have been identified (Table 2).

View larger version (32K): [in this window] [in a new window]   Figure 1. Representative example of TIMP-3 MSP assay. Methylation analysis of TIMP-3 promoter region CpG island in human PETs (T) and corresponding normal tissue (N). Lane U, Amplified product with oligonucleotides recognizing unmethylated TIMP-3 sequences. Lane M, Amplified product with oligonucleotides recognizing methylated TIMP-3 sequences. Normal tissue (N) and peripheral blood lymphocytes (PBL) as negative controls are completely unmethylated, whereas the in vitro methylated DNA (IVD) was used as a positive control for methylation. H2O, Water blanks. The used DNA size standard is a 50-bp ladder.

View larger version (84K): [in this window] [in a new window]   Figure 2. Representative examples of TIMP-3 immunohistochemistry. A, Gastrinoma 167/98T (arrowheads) that is characterized by hypermethylation of TIMP-3 does not express the TIMP-3 protein. In contrast, normal pancreatic islet cells (arrows) as internal positive control reveal strong expression of TIMP-3. B, Insulinoma 90 revealing no aberrant promoter methylation of TIMP-3 shows strong TIMP-3 expression. C, NNPC 155/98 with promoter hypermethylation of TIMP-3 has no detectable TIMP-3 immunoproduct. Magnification, -400; counterstain with hematoxylin.

View larger version (18K): [in this window] [in a new window]   Figure 3. Expression analysis of TIMP-3 by RT-PCR in human PETs. Expression of ß-actin was performed for all samples to ensure the integrity of the RT reactions. Lanes 1–16 contain RT-PCR products of total RNA from tumor tissues and control samples for TIMP-3 (648 bp) or ß-actin (778 bp) mRNA expression, respectively. Lane 1, 155/98T; 2, 166/98T; 3, 172/98T; 4, normal pancreatic islet cells; 5, normal kidney as a positive control; 6, 122T; 7, 164/98T; 8, 167/98T; 9, 74/96T; 10, 141/98T; 11, SW15122T; 12, positive control; 13, 26/96T; 14, 90T; 15, negative control for DNA contamination; and 16, water blank. M, DNA size standard (50-bp ladder).

Analysis of the clinical-genetic data revealed that PETs with lymph node and/or liver metastasis had a significantly higher rate of TIMP-3 alterations (11/14, 79%) than PETs without metastases (1/7, 14%; P < 0.02).

	Discussion

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As shown previously, PETs reveal allelic losses of 31% to 59% at chromosomal band 22q12.3 (17, 18, 19, 20). TIMP-3, colocalized with these markers, therefore appears to be an attractive candidate gene for genetic analysis in PETs to clarify its role for the tumorigenesis of these neoplasms. TIMP-3 is a member of the family of secreted inhibitors that blocks the activity of MMPs, therefore regulating matrix composition, cell growth, invasion, and migration, and has been shown to be implicated in tumor progression, including the development of metastasis (21, 22, 23, 24, 25, 30). A significant finding of our study is that methylation-associated silencing of TIMP-3 is prevalent in 44% of human PETs, which corresponds to loss of TIMP-3 protein expression. This effect is tumor-specific in the majority of PETs, because this epigenetic change was restricted to tumors in 17 of 18 matched normal/tumor pairs. Only the corresponding pancreatic tissue of vipoma 20/96 showed a weak methylated vs. a strong unmethylated MSP product. Low frequencies of methylation of the TIMP-3 promoter in corresponding normal tissue have also been described for some lung cancers (31) and may be due to contamination with adjacent tumor tissue. Methylation-associated loss of TIMP-3 expression was also identified in 78% of kidney, 27% of colon, and 19% of lung cancers (26). Notably, normal islet cells of the pancreas showed a strong level of TIMP-3 expression. It is, therefore, conceivable that TIMP-3 expression may be critical for the normal growth of these cell populations. The high frequency of promoter methylation in PETs may provide a mechanism for loss of such control, further indicating a tumor suppressor role for TIMP-3, as recently suggested (26, 31, 32).

However, the epigenetic phenomenon of promoter hypermethylation is the major mode for loss of TIMP-3 expression in PETs and seems not to be associated with LOH on 22q12.3, because four tumors with LOH and four tumors without LOH revealed this alteration. Thus, TIMP-3 might not be the main target of the relatively high frequency of allelic losses, suggesting the presence of another tumor suppressor gene in this region. Concordantly, homozygous deletions of TIMP-3, another important mechanism of tumor suppressor gene inactivation, were detected in only one tumor (166/98). The PCR-based detection of homozygous deletion is complicated in primary tumor specimens by the presence of admixed nonneoplastic tissue. We therefore minimized contamination from nonneoplastic tissue by microdissecting the tumors. Although we achieved a neoplastic cellularity of at least 85% in all tumor samples, the remaining nonneoplastic cell fraction (up to 15%) might have precluded a valid assessment of homozygous deletions in some of our cases. Intragenic mutations are another major mode of tumor suppressor gene inactivation but have not yet been described in TIMP-3, despite some germline mutations causing Sorsby fundus dystrophy (33, 34). We could not detect any TIMP-3 mutations screened by SSCP in 21 PETs. SSCP analysis as used in this study has a sensitivity of 80% for the identification of mutations in PCR-generated fragments (11, 35, 36). However, certain mutations, especially large intragenic deletions may not be detected using this method. This might be the case for the three tumors with loss of TIMP-3 expression but no detectable mutation or hypermethylation. In addition, other more distantly located regulatory elements may be sites of pathological mutation, or another gene product that regulates TIMP-3 expression might be affected. Nevertheless, intragenic mutational inactivation of TIMP-3 seems to be an infrequent event in PETs as it has been shown for the p16^INK4a tumor suppressor gene (8, 9, 10).

The only unequivocal criteria of malignancy in PETs are metastases or infiltrating growth in adjacent tissues. The availability of molecular markers for the distinction between benign and malignant forms of PETs would have great clinical relevance as a predictor of prognosis. Some previous studies suggested that allelic losses of chromosomes 1, 3p, 10q, 11q13, and 22q are associated with a more aggressive phenotype in PETs (12, 13, 14, 16, 20), whereas other groups could not confirm these associations (37, 38). In the present study, TIMP-3 alterations seem to be associated with a more aggressive tumor phenotype, because alterations occurred significantly more frequently in PETs with metastases than in PETs without metastases (79% vs. 14%; P < 0.02). This clinical association may support the recent hypothesis that reduced expression of TIMP-3 contributes to the metastatic process (22, 23, 24, 25), as it has been shown for gastric cancer (32). In the future, carefully designed, controlled, large-scale multicenter studies are required to further evaluate this potential prognostic association.

In summary, the presented data indicate that methylation-associated inactivation of TIMP-3 is one of the most common genetic events yet identified in PETs. This finding should stimulate further research using this tumor type as a model system to investigate the potential tumor suppressive function of TIMP-3, helping to refine our picture of the role TIMP-3 is playing in tumorigenesis. Large-scale multicenter studies are needed to further evaluate whether TIMP-3 alterations may be a useful prognostic marker for PETs.

	Acknowledgments

 
We thank all of the patients who participated in the study. The technical assistance of Ms. Karin Münch and the support of Dr. Marcus Schuermann are highly appreciated.

	Footnotes

 
This study was supported by the Deutsche Krebshilfe (Grant 10-1674-Ba I).

Abbreviations: LOH, Loss of heterozygosity; MMP, matrix metalloproteinases; MSP, methylation-specific PCR; NNPC, nonfunctioning neuroendocrine pancreatic carcinoma; PET, pancreatic endocrine tumor; SSCP, single-strand conformational polymorphism; TIMP-3, tissue inhibitor of metalloproteinase-3.

Received July 3, 2002.

Accepted December 11, 2002.

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