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Original Article |
Center for Molecular Medicine and Division of Endocrinology and Metabolism, University of Connecticut School of Medicine (T.M.S., J.C., M.B., A.A.), Farmington, Connecticut 06030; Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (R.T.J.), Bethesda, Maryland 20892; and Gastrointestinal Unit, Massachusetts General Hospital (D.C.C.), Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Andrew Arnold, M.D., Center for Molecular Medicine, University of Connecticut School of Medicine, 263 Farmington Avenue, Farmington, Connecticut 06030-3101. E-mail: . aarnold{at}nso2.uchc.edu
Abstract
Based upon molecular allelotyping and comparative genomic hybridization studies, chromosome 15q is the likely location of a tumor suppressor gene important in the pathogeneses of sporadic enteropancreatic endocrine tumors and parathyroid adenomas. Interest has focused on Smad3 as a candidate endocrine tumor suppressor gene because 1) it is localized to 15q and 2) it encodes a TGFß signaling molecule that has been identified as a binding partner of the multiple endocrine neoplasm type 1 gene product menin, itself involved in enteropancreatic and parathyroid neoplasia. To determine whether Smad3 plays a primary role in development of these tumors, 20 enteropancreatic tumors and 67 parathyroid adenomas were investigated for loss of heterozygosity at DNA markers surrounding Smad3. Twenty percent of enteropancreatic tumors and 24% of parathyroid adenomas showed loss. All 9 coding exons and intron-exon boundaries of the Smad3 gene were then sequenced in genomic DNA from all 20 enteropancreatic and 25 parathyroid tumors, including every case with loss of heterozygosity. No acquired clonal mutations, insertions, or microdeletions in Smad3 were detected in any tumors. Because inactivating somatic mutation is the hallmark of an authentic tumor suppressor, Smad3 is unlikely to function as a classical tumor suppressor gene in the pathogenesis of sporadic parathyroid or enteropancreatic endocrine tumors.
THE MULTIPLE ENDOCRINE neoplasia type 1 (MEN1) syndrome, an autosomal dominantly inherited disorder, is characterized by multiple tumors of the parathyroids, endocrine pancreas/gastrointestinal tract, and anterior pituitary. Affected individuals bear heterozygous inactivating germline mutations of the MEN1 tumor suppressor gene located on chromosome 11q13 (1, 2, 3). Acquired inactivation of both MEN1 alleles, consistent with the two-hit model for classical tumor suppressor genes (4, 5, 6), plays a role in the pathogenesis of sporadic enteropancreatic tumors and parathyroid adenomas as well (7, 8, 9, 10, 11). Menin, the protein product of the MEN1 gene, has virtually no homology to other known proteins, with only a nuclear localization signal sequence at the carboxyl terminus providing a clue to its function (12). Proteins that interact with menin have been sought in hopes that this information would help elucidate menin’s functions. Recently, it was reported that menin can directly interact with Smad3, a downstream component of the TGFß signaling pathway (13). When TGFß receptors are activated by their ligand, Smad3 becomes phosphorylated and can subsequently enter the nucleus to alter transcription, usually leading to an inhibition of cell growth (reviewed in Refs. 14 and 15). However, when menin levels were decreased by antisense MEN1 gene transcripts, pituitary cells became less susceptible to the growth inhibitory effects of TGFß. Antisense menin could also inhibit the transcription of TGFß target genes (13). These results suggest that menin, through its effect on Smad3, may play an important role in the growth inhibitory actions of the TGFß pathway, and the loss of functional menin that occurs in MEN1 patients and in sporadic endocrine tumors may decrease the growth restriction exerted by this pathway.
Chromosome 15, the location of Smad3, has been implicated as the site of a still-unidentified tumor suppressor gene in both sporadic enteropancreatic tumors and parathyroid adenomas. Frequent deletions of murine chromosomes 9 and 16 were found in a mouse model of pancreatic endocrine tumors (16), suggesting that the syntenic regions of the human genome, including chromosome 15q, may harbor important tumor suppressor genes for the corresponding human disease (17). This concept was reinforced by genome-wide allelotyping studies that revealed recurrent losses of 15q in human pancreatic endocrine tumors (18). Studies of sporadic parathyroid adenomas using both molecular allelotyping and comparative genomic hybridization have also revealed highly recurrent clonal losses on chromosome 15 (19, 20, 21, 22), and Smad3 is located in this region.
Finally, interest in Smad3 as a compelling candidate parathyroid and enteropancreatic tumor suppressor gene has intensified because, in addition to its suitable genomic location and interactions of Smad3 with menin, 1) Smad3 interacts with the vitamin D receptor (VDR) and can serve as a transcriptional coactivator of the VDR (23, 24), suggesting that Smad3 may play additional roles in regulating the growth of the parathyroid glands through its actions in this growth inhibitory pathway (25, 26); and 2) Smad4 has been implicated in enteropancreatic tumors (27), underscoring an important role for TGFß signaling in enteropancreatic tumors.
Proof that Smad3 is indeed a classic parathyroid and/or enteropancreatic tumor suppressor gene would require the demonstration of somatic mutations inactivating both Smad3 alleles in a subset of tumors; frequently, one allele is lost through a sizeable deletion or loss of heterozygosity (LOH) event, and the remaining allele contains a specific inactivating mutation (4). Therefore, we sought such critical deletions and internal mutations in a rigorous study of Smad3’s potential role as a primary driver of parathyroid and enteropancreatic tumorigenesis.
Subjects and Methods
Patient and tumor samples
Parathyroid adenoma samples were collected from patients undergoing parathyroidectomy of the management for primary hyperparathyroidism. All patients were surgically and pathologically proven to have single gland disease with no malignant features. Patients had no previous history of head and neck irradiation and no family history of MEN syndromes or familial hyperparathyroidism syndrome.
Pancreatic endocrine tumors were obtained from patients postoperatively and were classified as functional or nonfunctional on the basis of their clinical presentation and/or serum hormone levels. All tumors were sporadic; patients had no family history of MEN syndromes or von Hippel-Lindau syndrome.
Upon surgical removal, tissue was dissected and frozen in liquid nitrogen. Genomic DNA was extracted from the tissue by proteinase K digestion, followed by phenol-chloroform extraction and ethanol precipitation. Control normal DNA was extracted from peripheral blood leukocytes from the same patients. Samples were obtained with patient consent in accordance with institutional review board guidelines.
LOH analysis
Sixty-seven sporadic parathyroid adenomas and 21 pancreatic endocrine tumors were examined for LOH using chromosome 15 microsatellite polymorphic markers in the region of Smad3 according to the University of California-Santa Cruz Human Genome Project Working Draft (http://genome.ucsc.edu): D15S153, D15S988, and D15S131. Fluorescently labeled primers (PE Applied Biosystems, Inc., Foster City, CA) specific for each microsatellite were used to amplify genomic DNA from tumor and corresponding peripheral blood leukocytes or other normal tissue by PCR. Primers were multiplexed in a single 15-µl reaction. PCR conditions were as recommended by the manufacturer. Samples were electrophoresed on an ABI PRISM 377 sequencer, and data were analyzed using Genescan and Genotyper software (PE Applied Biosystems, Inc.). To account for differences in gel loading and PCR amplification efficiency, a ratio of the heights of the allele peaks in the tumor and the normal samples was calculated using the following formula: (peak 1 height tumor/peak 2 height tumor)/(peak 1 height normal/peak 2 height normal). A value of more than 2.0 or less than 0.5, representing a loss of intensity in the tumor of 50% of 1 of the 2 alleles present in the normal tissue, was scored as LOH.
Smad3 sequencing
Twenty-five parathyroid adenomas, including all 16 that showed 15q LOH and an additional 9 that were informative for markers flanking the Smad3 gene and clearly showed no loss, and all 20 enteropancreatic endocrine tumors consisting of 4 gastrinomas, 10 insulinomas including 1 recurrence, 1 glucagonoma, 1 PTHrP-producing pancreatic endocrine tumor, and 4 nonfunctioning pancreatic endocrine tumors, were analyzed for Smad3 mutations. Each of the 9 Smad3 exons (28) was amplified from 25 ng genomic DNA using PCR. Primers used for PCR and sequencing and PCR conditions are detailed in Table 1
. PCR reactions included 25 ng DNA, 20 pmol of each primer, 200 µM of each NTP, 1.25 U AmpliTaq Gold DNA polymerase (PE Applied Biosystems, Inc.), PCR buffer (PE Applied Biosystems, Inc.), and MgCl2 (concentrations given in Table 1) in a 20-µl reaction. Thermocycling consisted of an initial denaturation for 95 C for 10 min; 35 cycles of 95 C for 30 sec, 55–60 C for 30 sec, and 72 C for 30 sec; and a final extension at 72 C for 10 min.
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Results
LOH on chromosome 15 has been reported in subsets of parathyroid adenomas and enteropancreatic endocrine tumors. To establish which samples in this study have loss in the region surrounding Smad3 and thereby highlight tumors especially likely to reveal mutations in the remaining allele of a classic tumor suppressor gene in the region, several microsatellite markers surrounding the candidate gene were analyzed. Four of the 20 enteropancreatic tumors, 1 glucagonoma, 1 PTHrP-producing pancreatic endocrine tumor, and 2 clinically nonfunctioning pancreatic endocrine tumors, were found to have LOH. All 20 enteropancreatic tumors were analyzed for mutations in the complete protein-coding region and intron-exon boundaries of Smad3. Neither the tumors with nor those without 15q LOH showed alterations consistent with deletions, insertions, or point mutations.
In a similar manner, 67 parathyroid adenomas were analyzed for LOH near Smad3. The results of this study in combination with those of previously published LOH and comparative genomic hybridization studies of some of the same tumors indicated that loss in the region including Smad3 was present in 16 adenomas (24%). The entire coding region of the Smad3 gene along with all intron-exon borders was then sequenced in the 16 samples with allelic loss. An additional 9 tumors that were informative (heterozygous) at markers flanking Smad3 and showed no LOH were sequenced to help address the related hypothesis that haploinsufficiency of Smad3 might contribute to the pathogenesis of parathyroid adenomas. No tumor-specific somatic mutations in Smad3 were detected, although a previously reported polymorphism (29) in exon 3 was found in 2 tumors that had shown LOH in the region surrounding Smad3 and in the corresponding normal control DNA from the same patients (Fig. 1). One patient’s constitutional DNA showed homozygosity for the polymorphism, whereas the other patient was heterozygous. In a Finnish study this polymorphism was found in 6.4% of the general population and did not segregate with cancer in families with hereditary nonpolyposis colon cancer (29).
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TGFß signaling is an important regulator of cell proliferation. Recent findings that menin interacts with Smad3, a downstream modulator of TGFß signaling, suggest that this pathway may play an important role in control of the growth of endocrine tissues. Depletion of menin in cultured cells, similar to what would occur in tumors of MEN1 patients or in sporadic tumors with biallelic disruption of MEN1, was shown by Kaji et al. (13) to disrupt normal TGFß signaling and lead to loss of the antiproliferative effects of TGFß. Disruption of Smad3, which would also disrupt TGFß signaling, might well lead to a neoplastic outcome in these tissues. This argument is supported by the finding that Smad4 mutations have been identified in a small group of enteropancreatic tumors (27). Of uncertain relevance to human neoplasia, colon (but not endocrine) tumors have been an inconsistent finding in reports of Smad3 knockout mice (30, 31, 32). Interestingly, Smad3 not only links menin to TGFß signaling, but also to the vitamin D transcriptional regulatory pathway (23). 1,25-Dihydroxyvitamin D3, which binds the VDR and allows VDR to alter transcription in a cell, is also a negative regulator of parathyroid growth (25, 26), Therefore, Smad3 could serve as a crucial negative regulator of cell growth in parathyroid glands through a TGFß/menin pathway and by interacting with the VDR. For these biologically based reasons plus the intriguing location of Smad3 in a chromosomal region recurrently showing tumor-specific LOH in tumor types associated with the MEN1 syndrome, Smad3 emerged as an important candidate tumor suppressor gene in parathyroid adenomas and enteropancreatic tumors.
We fully sequenced the protein-coding sequence and intron-exon junctions of the Smad3 gene in 20 enteropancreatic endocrine tumors and 25 parathyroid adenomas. One polymorphism, but no clonal somatic mutations, was detected. A strength of this study is that both tumors with and without LOH in the Smad3 region were included, thereby maximizing the chances of finding either hemizygous or biallelic internal inactivating mutations or mutations affecting only one allele paired with a normal allele (haploinsufficiency). Although there are potential alternative mechanisms of Smad3 gene inactivation, such as hypermethylation, promoter mutation, or intronic mutations that affect mRNA splicing or expression that would not have been detectable by this intron-exon junctional and coding region-based sequencing approach, it is exceedingly improbable that these mechanisms would inactivate Smad3 to the exclusion of mutations in the rigorously searched coding region of the gene (4, 5, 6). Indeed, every classic tumor suppressor gene with established importance in common nonfamilial forms of human neoplasia would have been identified by this approach (4, 5, 6). Therefore, it is unlikely that acquired biallelic or 2-hit inactivation/loss of function of Smad3 commonly plays a primary role in driving sporadic parathyroid or enteropancreatic endocrine tumorigenesis, i.e. Smad3 is not likely to be a classic tumor suppressor gene for these tumor types. These observations do not, however, exclude the possibility that secondary changes in Smad3 function, perhaps involving interaction with menin or established components of the TGFß signaling pathway, might be important in the dysregulation of these endocrine cell types.
Acknowledgments
Footnotes
This work was supported in part by The Dr. and Mrs. Solomon Rosenblatt Research Fund in Molecular Medicine, the AGA Miles and Shirley Fiterman Foundation Basic Research Award, and a Juvenile Diabetes Foundation Research Grant.
Abbreviations: LOH, Loss of heterozygosity; MEN1, multiple endocrine neoplasm type 1; VDR, vitamin D receptor.
Received February 20, 2002.
Accepted April 12, 2002.
References
This article has been cited by other articles:
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  E. H. Samander and A. Arnold Mutational Analysis of the Vitamin D Receptor Does Not Support Its Candidacy as a Tumor Suppressor Gene in Parathyroid Adenomas J. Clin. Endocrinol. Metab., December 1, 2006; 91(12): 5019 - 5021. [Abstract] [Full Text] [PDF]
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L. J. Krebs, T. M. Shattuck, and A. Arnold HRPT2 Mutational Analysis of Typical Sporadic Parathyroid Adenomas J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5015 - 5017. [Abstract] [Full Text] [PDF]
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E. M Pinto, A. E. C. Billerbeck, M. C. B. V. Fragoso, B. B. Mendonca, and A. C. Latronico Deletion Mapping of Chromosome 17 in Benign and Malignant Adrenocortical Tumors Associated with the Arg337His Mutation of the p53 Tumor Suppressor Protein J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2976 - 2981. [Abstract] [Full Text] [PDF]
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 T. M. Shattuck, S. Valimaki, T. Obara, R. D. Gaz, O. H. Clark, D. Shoback, M. E. Wierman, K. Tojo, C. M. Robbins, J. D. Carpten, et al. Somatic and Germ-Line Mutations of the HRPT2 Gene in Sporadic Parathyroid Carcinoma N. Engl. J. Med., October 30, 2003; 349(18): 1722 - 1729. [Abstract] [Full Text] [PDF]
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