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Missense Mutations of MADH4

Characterization of the Mutational Hot Spot and Functional Consequences in Human Tumors

Departments of ¹ Pathology, ² Oncology, ³ Surgery, and ⁴ Public Health, The Johns Hopkins Medical Institutions, Baltimore, Maryland, and ⁵ Temple University School of Medicine, Philadelphia, Pennsylvania

ABSTRACT

Purpose and Experimental Design: The mutational spectrum of MADH4 (DPC4/SMAD4) opens valuable insights into the functions of this protein that confer its tumor-suppressive nature in human tumors. We present the MADH4 genetic status determined on a new set of pancreatic, biliary, and duodenal cancers with comparison to the mutational data reported for various tumor types.

Results: Homozygous deletion, followed by inactivating nonsense or frameshift mutations, is the predominant form of MADH4 inactivation in pancreatic cancers. Among the naturally occurring MADH4 missense mutations, the MH2 domain is the most frequent target (77%) of missense mutations in human tumors. A mutational hot spot resides within the MH2 domain corresponding to codons 330 to 370, termed the mutation cluster region (MCR). A relationship was found between the locations of the missense mutations (the MH1 domain, the MH2-MCR, and the MH2 outside of the MCR) and the tumor types, suggesting environmental or selective influences in the development of MADH4 mutations. Immunohistochemical studies for Madh4 protein in nine archival cancers (six pancreatic cancers, two duodenal cancers, and one biliary cancer) with known missense mutations indicated that all mutations within the MH1 or MH2 domain COOH-terminal to the MCR (seven of nine cases) had negative or weak labeling, whereas two cancers with mutations within the MCR had strong positive nuclear labeling for Madh4 protein.

Conclusions: These findings have important implications for in vitro functional studies, suggesting that the majority of missense mutations inactivate Madh4 by protein degradation in contrast to those that occur within the MCR.

Introduction

Human pancreatic ductal adenocarcinomas inactivate the tumor suppressor gene MADH4 (DPC4, SMAD4) with a high frequency (1) . This inactivation occurs most commonly by homozygous deletion (HD), but some tumors may also inactivate the gene by loss of heterozygosity (LOH) coupled with a mutation in the remaining allele. Inactivation by nonsense mutation may cause the loss of protein expression by enhanced proteosomal degradation (2 , 3) . Even when expressed as protein, missense mutations may result in loss of a specific function of the Madh4 protein such as DNA binding or Smad protein interactions (2, 3, 4, 5, 6, 7, 8, 9) . Thus, the location of these mutations can provide clues to key structural features that mediate the tumor-suppressive function of MADH4.

Members of the Smad protein family, including Madh4, have two evolutionarily conserved regions termed the MH1 and MH2 domains (Mad homology 1 and 2). The NH₂-terminal MH1 domain (codons 1 through 142) is responsible for sequence-specific DNA binding (5, 6, 7 , 10, 11, 12) , whereas heteromerization and transactivation functions have been largely attributed to the MH2 domain [codons 319–552 (6 , 8 , 13) ]. In addition, the MH2 region has been shown to partially interfere with the DNA-binding function of the MH1 region (5 , 7 , 8 , 12 , 14) . These domains are separated by a linker region that contains a 48-amino acid segment called the Smad4 activation domain required for the activation of Smad4-dependent signaling responses (15) .

In the seminal study by Hahn (1) , six pancreatic carcinomas were found to have intragenic mutations of the MADH4 gene, only one of which was a missense mutation. Schutte et al. (16) reported additional missense mutations in a pancreatic cancer cell line and an ovarian carcinoma. Missense mutations have since been reported in biliary cancers (17) , neuroendocrine tumors (18) , colorectal cancer (19, 20, 21, 22, 23) , juvenile polyposis syndrome (24, 25, 26, 27, 28) , ovarian cancer (29) , and lung cancers (30) . Thus, the mapping of these mutations to the known domains of the MADH4 gene and their presumed effect on the function of the Dpc4 protein are in need of update. We present new data characterizing the mutations of the MADH4 gene in a large series of additional pancreatic cancer xenografts and cell lines. These combined data help to better define the spectrum of mutations in the MADH4 gene with respect to clustering and potential effects on tumor-suppressive function in pancreatic cancers. We compare our findings with those reported for other human tumor types in an effort to consider the possible association between the mutation location and tumor types.

Materials and Methods

Generation of Xenografts and Cell Lines.
The generation of xenografted tumors derived from pancreatic, biliary, and other tumor types has been described in detail previously (31) . Human pancreatic cancer cell lines Panc 9.06, Panc 8.13, Panc 3.27, Panc 2.8, and PL45 are low-passage pancreatic carcinoma cell lines kindly provided by Dr. Elizabeth Jaffee (32) or described previously (33) . All were recently made available through the American Type Culture Collection (Manassas, VA). All cell lines were cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin). Cells were incubated at 37°C in a humidified atmosphere of 5% CO₂ in air.

Determination of LOH.
Previous work demonstrated a high frequency of LOH at 18q21.1 in these pancreatic cancer xenografts by use of dinucleotide markers, described previously in detail (34) .

Amplification and Sequencing of Exons 0–11 of MADH4.
PCR amplification of exons 0–11 from genomic DNA was performed as described previously (16 , 35) . PCR-amplified products were purified using QIAquick (Qiagen) and studied by automated sequencing using nested primers and an ABI Prism model 3700 (Applied Biosystems, Foster City, CA). Sequence analysis used Sequencher version 4.0.5 software (Gene Codes, Ann Arbor, MI). Verification of the mutation was accomplished by sequencing of a second PCR product derived independently from the original template.

Immunohistochemistry.
Unstained 5-µm sections were cut from the archival paraffin blocks of eight pancreatic, biliary, or duodenal cancers with tumorigenic missense mutations of the MADH4 gene. Paraffin blocks corresponding to one of the duodenal carcinomas having a missense mutation were unavailable. For this case, samples of the normal duodenal mucosa and xenografted tumor were fixed overnight in 10% buffered formalin and embedded in paraffin, and sections were cut. Slides were deparaffinized by routine techniques followed by incubation in 1x sodium citrate buffer (diluted from 10x heat-induced epitope retrieval buffer; Ventana-Bio Tek Solutions, Tucson, AZ) before steaming for 20 min at 80°C. Slides were cooled for 5 min and incubated with a 1:100 dilution of monoclonal antibody to Madh4 protein (clone B8; Santa Cruz Biotechnology, Santa Cruz, CA) using the Bio Tek-Mate 1000 automated stainer (Ventana-Bio Tek Solutions). This antibody has previously been shown to be a sensitive and specific marker of MADH4 genetic status in pancreatic cancers (36) . Anti-Madh4 antibody was detected by secondary antibody, followed by avidin-biotin complex and 3,3'-diaminobenzidine chromagen. Sections were counterstained with hematoxylin and evaluated by two of the authors (C. A. I-D. and S. E. K.), with agreement in all cases.

Protein Structure Analysis.
The crystal structures of the Smad3 MH1 domain bound to the Smad binding element, and the Smad4 MH2 domains were obtained from the National Center for Biotechnology Information Structure database.⁶ Cn3D software version 4.1, also available from this web site,⁶ was used to visualize the protein domains and to identify the positions corresponding to missense mutations in these structures.

Statistical Analysis.
The location of missense mutations [MH1, mutation cluster region (MCR), or COOH-terminus of the MH2] in relation to the primary tumor type was determined by ² analysis. Values of 0.05 were considered significant.

Results

MADH4 Gene Inactivation in Pancreatic Cancer.
We analyzed the MADH4 gene in 63 new cases representing 54 pancreatic cancer xenografts, 2 duodenal cancer xenografts, 2 biliary cancer xenografts, and 5 pancreatic cancer cell lines. LOH at 18q21 was determined to be present for all but one of the duodenal cancers. Homozygous deletion was found in 13 cases (10 xenografts and 3 cell lines of pancreatic cancer). Ten of these HDs involved the entire coding region of MADH4. In three additional cases, only a portion of the coding region was homozygously deleted, corresponding to exons 7–11 in cell line Panc 8.13, exons 5–11 for xenograft PX191, and exons 1–4 for xenograft PX194. In all 13 cases, the presence of a HD was confirmed by the failure of PCR to amplify contiguous DNA segments in the presence of appropriate positive controls.

For the 50 cases in which no HD was found, exons 0–11 of the MADH4 gene were PCR amplified and sequenced. Eighteen different intragenic mutations in 17 cases were identified, corresponding to 13 pancreatic cancer xenografts, two duodenal cancer xenografts, one biliary cancer xenograft, and one pancreatic cancer cell line (Table 1) . In four pancreatic cancer xenografts and the cancer cell line, an insertion/deletion mutation was found, and each was predicted to cause a frameshift in the coding sequence. In three additional pancreatic cancer xenografts, a nonsense mutation was found, and in nine cases (six pancreatic cancer xenografts, two duodenal cancer xenografts, and one biliary cancer xenograft), a missense mutation was identified in the retained MADH4 allele. One of the missense mutations in a pancreatic cancer xenograft occurred in a location predicted to cause a splice site alteration at exon 8. One of the duodenal cancer xenografts (PX255) contained two different mutations, a missense mutation of codon 361 resulting in replacement of an Arg with a Trp residue at that site, and a nonsense mutation of codon 445 resulting in replacement of an Arg residue with a termination signal. This latter xenograft was the one tumor lacking LOH. In all cases, intragenic mutations were confirmed by sequencing a second PCR product independently generated from the original template DNA.

Mapping of Mutations and Identification of the Mutational Hot Spots of MADH4.
Forty-two pancreatic cancer xenografts and 13 pancreatic cancer cell lines have previously been analyzed for genetic alterations of MADH4 and reported in detail (1 , 16) . Together with the current data, MADH4 inactivation was found in a total of 60 of 114 pancreatic cancer xenografts or cell lines (53%), of which 38 (63%) are due to HD, 14 (23%) are due to nonsense or frameshift mutation, and 8 (13%) are due to a missense mutation.

Table 2 summarizes the missense mutations within MADH4 in human neoplasms and syndromes reported in the National Center for Biotechnology Information PubMed database⁷ to date. A total of 70 reported missense mutations including this current study affect 51 unique codons. Missense mutations affecting eight codons in particular have each been reported in more than one study. These comprised codon 118, reported in this study in a pancreatic cancer xenograft (PX133) and in a colorectal cancer (21) , codon 350 in a small intestinal cancer (37) and a colorectal cancer (21) , codon 386 in syndromic juvenile polyps (28) and in an ovarian cancer (29) , codon 433 in a biliary cancer (17) and a colorectal cancer (22) , and codon 523 in a biliary cancer (17) and a pancreatic cancer (38) . Missense mutations affecting codons 330 and 355 were each reported in three different studies. All three codon 330 mutations were in a colorectal cancer (21 , 22 , 39) , and codon 355 mutations occurred in a pancreatic cancer cell line (40) and in two colorectal cancers (20 , 39) . Mutations affecting codon 351 were reported in five independent studies representing two colorectal cancers (19 , 23) , one small bowel cancer (37) , and two ovarian cancers (16 , 29) . Missense mutations affecting codon 361 were reported in three colorectal cancers (22 , 41 , 42) , syndromic juvenile polyps (25, 26, 27) , and a small intestinal cancer (37) in addition to a duodenal cancer xenograft reported in the current study (PX255).

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Distribution of mutations reported for the MADH4 gene. The location of all missense mutations is indicated in reference to the MADH4 coding sequence. Arrow colors indicate the human tumor types from which the mutations are reported. Green, pancreatic cancer; pink, duodenal cancer; dark blue, sporadic colorectal cancer; light blue, juvenile polyposis syndrome; brown, small intestinal cancer; gray, acute myelogenous leukemia; yellow, biliary cancer; orange, hepatocellular cancer; red, ovarian cancer. Black arrows are mutations reported in MADH4 but not validated by sequencing of an independent amplification product. The coding regions corresponding to amino acids 330–370 are indicated by a bracket to illustrate the mutation cluster region of the MH2 domain. This distribution of mutations is significantly associated with tumor type (P < 0.001).

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Distribution of missense mutations within functional domains of the highly similar Smad3 MH1 region or the Madh4 MH2 region. Only validated mutations are represented (as defined in Table 2 ). A, crystal structure of two molecules of the Smad3 MH1 protein domain bound to DNA. Smad3 MH1 protein is shown in blue and purple, and DNA is shown in green and brown. The NH2 terminus of the structure is noted as well as the approximate locations of the double loop, basic helix, and ß-hairpin motifs. Amino acid residues targeted by missense mutations in MADH4 are indicated in yellow. B and C, crystal structure of the MadH4 MH2 domain. The protein is shown in purple, and the amino acid residues targeted by missense mutations are indicated by yellow. In B, missense mutations corresponding to the mutation cluster region are shown, and in C, missense mutations corresponding to the COOH end of the MH2 are shown.

Relationship of Madh4 Protein Expression to Location of Missense Mutation.
Homozygous deletions and nonsense mutations of MADH4 are thought to result in loss of protein expression or an unstable protein that may be targeted for degradation (2 , 36) . We determined the status of Madh4 protein expression in nine cancers (six pancreatic cancers, two duodenal cancers, and one biliary cancer) in which MADH4 contained a missense mutation (Fig. 3) . Four cases contained a mutation within the MH1 domain, and five contained a mutation within the MH2 domain, two of which corresponded to the MCR.

View larger version (88K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Immunohistochemical detection of Madh4 protein in nine human cancers with known MADH4 missense mutations. Shown are four cancers with a missense mutation within the MH1 domain (top panels) and five cancers with a missense mutation within the MH2 domain (bottom panels). The codon affected by the mutation and the resultant amino acid changes are listed for each case. The presence or absence of Madh4 protein expression is seen to correlate with the location of the missense mutations. Within the MH1, mutations at codons 86, 99, and 127 result in loss of protein expression within the neoplastic epithelium, in contrast to the positive labeling of stromal cells. Madh4 protein is detectable in the case of mutation at codon 118, although labeling is weak and apparently confined to the cytoplasm. Mutations within the mutation cluster region of the MH2 domain (codons 353 and 361) have strong nuclear labeling in each case, whereas mutations outside of the cluster region at the 3' end of the gene weakly label (codon 492) or have complete loss of Madh4 protein (codons 493 and 499).

Discussion

Homozygous deletion is the most common form of MADH4 inactivation. In these instances, the loss of tumor-suppressive function is clearly mediated by the loss of Madh4 protein expression. A similar phenomenon may also occur in human tumors where a nonsense, splice site, or frameshift mutation occurs; Madh4 protein is immunohistochemically undetectable, a finding highly concordant with the MADH4 genetic status (36) . In these examples, gross splicing or translation errors are expected to preclude translation or result in an unstable protein product that is targeted for degradation (2 , 44) .

In contrast to MADH4 inactivation caused by HD or nonsense type mutations, the in vivo functional consequences of missense mutations have not been explored in detail. Our data indicate that the majority of missense mutations may also inactivate MADH4 through enhanced degradation of an unstable protein product. Specifically, we have noted that many missense mutations target structural residues, in particular those mutations occurring in the MH1 or COOH end of the MH2 domains. The decreased stability of missense mutations occurring in the MH1 domain has been demonstrated in vitro (3 , 5) , where the protein is thought to be rapidly degraded in vivo by the ubiquitin-proteasome pathway (44) . Our data indicate that missense mutations occurring COOH-terminal of the MH2 domain may also be targeted for rapid degradation because Madh4 protein expression is undetectable or at most weakly positive in pancreatic cancers with missense mutations in this region. In the prior studies by Wilentz et al. (36 , 45) characterizing MadH4 protein expression in relation to MADH4 genetic status, no cancers with missense mutations were available for study. We are aware of only two other studies in which MADH4 missense mutations have been correlated to immunohistochemical labeling for Madh4 (24 , 38) . Interestingly, both studies also reported loss of Madh4 protein expression in tumors with missense mutations COOH-terminal of the MCR and support the concept that this coding region is also exquisitely sensitive to structural changes introduced by single missense mutations.

Our data also indicate that a MADH4 MCR exists for naturally occurring missense mutations and that carcinomas with missense mutations within this MCR can retain Madh4 protein expression. The MCR, spanning codons 330–370, corresponds to the loop/helix structure of the MH2 domain, supporting its functional significance proposed from structural studies (6 , 46 , 47) . The loop/helix structure is believed necessary for heterocomplex formation of Madh4 with other Smad proteins and translocation into the nucleus, where it associates with other transcription factors and regulates expression of ligand-responsive genes (9) . Our data indicate that Madh4 mutations occurring within the MCR need not affect protein stability or nuclear localization because two duodenal carcinomas with mutations in this region had strong nuclear labeling of Madh4 in the neoplastic epithelium. Thus, mutations within this region may have less of an impact on protein structure and/or function than found for mutations in other Madh4 domains.

Our findings also indicate that the mutational spectrum observed for MADH4 differs according to human tumor type. For example, missense mutations within pancreatic cancers predominantly occur within the MH1 domain or the MH2 domain COOH-terminal to the MCR, whereas those mutations affecting gastrointestinal mucosa (small bowel or colorectal carcinomas) occur more frequently within the MCR. Tumor type clustering of mutations as a function of environmental influences is a recognized phenomenon of other genes known to be important in carcinogenesis, such as p53 and K-ras (48 , 49) . Thus, differential influences may exist that contribute to the mutational spectrum of MADH4.

Our findings have obvious implications for in vitro functional studies that presume the effective protein expression of Madh4 in human tumors with missense mutations of the gene. The accumulated data on MADH4 missense mutations now indicate that protein instability, not loss of a specific function, is the consequence of the majority of mutations identified to date. In this context, most missense mutations can be thought of as obliterative mutations, functionally analogous to HDs and nonsense mutations. However, the presence of a distinct MCR whose mutations permit continued Madh4 protein expression and successful nuclear localization indicates alternative properties of Madh4 targeted in these tumors.

FOOTNOTES

Grant support: Supported by the NIH Specialized Programs of Research Excellence in Gastrointestinal Cancer Grant CA 62924 and Grant CA 68228.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Scott E. Kern, Department of Oncology, Room 461, Cancer Research Building, 1650 Orleans Street, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231. Phone: (410) 614-3316; Fax: (410) 614-9705; E-mail: sk{at}jhmi.edu

⁶ www.ncbi.nlm.nih.gov/Structure.

⁷ http://www.ncbi.nlm.nih.gov/PubMed.

Received 7/ 8/03; revised 12/ 8/03; accepted 12/ 8/03.

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