Journal of Surgical Research 121, 50 –55 (2004) doi:10.1016/j.jss.2004.03.008 The Unsolved Enigma of CDH1 Down-Regulation in Hereditary Diffuse Gastric Cancer Paola Concolino,* Valerio Papa,† Simona Mozzetti,* Cristiano Ferlini,* ,1 Fabio Pacelli,† Enrica Martinelli,* Riccardo Ricci,‡ Flavia Filippetti,* Giovanni Scambia,* ,§ and Giovanni Battista Doglietto† *Laboratory of Antineoplastic Pharmacology, †Department of Digestive Surgery, and ‡Department of Pathology, Università Cattolica Sacro Cuore, Rome, Italy; and §Department of Oncology, Università Cattolica Sacro Cuore, Campobasso, Italy Submitted for publication December 16, 2003 mechanisms of CDH1 suppression are involved to explain CDH1 down-regulation in HDGC patients without CDH1 mutations and promoter methylation. © 2004 Background. Hereditary diffuse gastric cancer (HDGC) is a disease mediated by down-regulation of the tumor suppressor E-cadherin (CDH1). This disease is particularly dangerous because of the youth of the patients, and for clinical management, hampered by the submucosal spread of tumor invisible at endoscopy. Two mechanisms of CDH1 down-regulation have been described in HDGC: missense mutations in the CDH1 gene and gene silencing through promoter methylation. Materials and methods. Seven patients affected by HDGC were enrolled. Tumor tissues were checked for CDH1 expression by immunohistochemistry (IHC). CDH1 DNA sequencing was performed for all its 16 exons from tumor and normal tissues of the same patients to detect somatic and germ-line mutations. Methylation promoter study was performed using specific primers and PCR. Results. IHC analysis confirmed CDH1 downregulation in all patients. DNA sequencing revealed the presence of six missense mutations in five patients. Four mutations were at the EC-3 domain of CDH1, whereas the other two were found in the cytoplasmic region interacting with catenins. All six mutations were absent in normal tissue, thereby excluding its presence in germ-line cells. Four patients exhibited both DNA missense mutations and gene silencing through promoter methylation. In two patients we did not notice either DNA missense mutations or promoter methylation. Conclusion. CDH1 somatic mutations and promoter methylation synergistically induce CDH1 downregulation in HDGC patients, whereas germ-line mutations are relatively rare. However, other unknown Elsevier Inc. All rights reserved. Key Words: CDH1; hereditary diffuse gastric cancer; promoter methylation; DNA mutations. INTRODUCTION Gastric cancer is a disease highly prevalent throughout the world and one of the leading causes of death. The majority of gastric cancer is sporadic in nature. In addition to the sporadic form, a small percentage of gastric cancer (1–3%) arises as a result of clearly identified inherited gastric cancer predisposition syndromes [1–3]. Such familiar cancers frequently display the diffuse gastric cancer histotype, with an autosomal dominant pattern of inheritance [4]. This disease is referred to as hereditary diffuse gastric cancer (HDGC). It is particularly dangerous because the disease occurs at a young age and the majority of tumors spread submucosally rather than forming a visible exophytic mass, thereby affecting the efficacy of clinical surveillance through current endoscopic methods. Recently, linkage analysis disclosed the molecular target underlying the vast majority of HDGC. In fact, heterozygotes carrying a mutation at the E-cadherin (CDH1) level have a high risk of developing such a disease with poor prognosis and a 5-year survival rate of only 10% [4]. An increased risk to develop sporadic diffuse gastric cancer has been assigned to some CDH1 polymorphisms in the promoter and coding region [5], but no univocal data have been obtained in diverse populations [6], so that the pathogenetic role of CDH1 in sporadic diffuse gastric cancer is still uncertain. CDH1 is a glycoprotein with a large extracellular 1 To whom correspondence and reprint requests should be addressed. E-mail: cferlini@rm.unicatt.it. 0022-4804/04 $30.00 © 2004 Elsevier Inc. All rights reserved. 50 CONCOLINO ET AL.: HEREDITARY DIFFUSE GASTRIC CANCER domain comprising five cadherin-motif subdomains, a single-pass transmembrane segment, and a short conserved cytoplasmic domain, which interacts with several proteins collectively referred to as catenins [7]. CDH1 is involved in the cell-to-cell adhesion process and plays a prominent role in epithelial differentiation and in the maintaining of the polarized feature of selected epithelial cells, such as in the case of gastric epithelial tissue. Experimental models support a role for CDH1 as a potent invasion/tumor suppressor in HDGC as well as in lobular breast carcinoma [8]. At diagnosis of HDGC, genetic counseling is required for all the relatives of patients susceptible to the disease. If through genetic analysis young asymptomatic carriers of CDH1 mutations are identified, prophylactic total gastrectomy should be considered as the unique tool to prevent the insurgence of a fatal disease due to the lack of effective chemopreventive strategies and the above-mentioned poor efficiency of clinical surveillance [9, 10]. However, following this approach, recent data have demonstrated the presence of an alternative mechanism of CDH1 suppression in the absence of DNA mutation in germ-line cells. This mechanism is epigenetic and is mediated by promoter methylation and the consequent silencing of the CDH1 gene [11]. Due to the absence of germ-line DNA mutations, in this case genetic tests are not useful in relatives of patients. The aim of this work was to evaluate what is the actual prevalence of genetic versus epigenetic mechanisms of CDH1 down-regulation. Results have shown that in our clinical setting DNA mutations were present only in tumor specimens, and absent in all the cases in germ-line cells. On the other hand, in the same setting, CDH1 down-regulation was often associated with CDH1 promoter hypermethylation. This fact suggests that epigenetic mechanisms and somatic mutations play a prominent role in inducing CDH1 downregulation, and that other unknown factors diverse from germ-line CDH1 mutations could frequently trigger HDGC. MATERIALS AND METHODS Patients The hospital records of 639 patients affected by primary gastric cancer who were consecutively admitted to our unit during the period 1981–1995 [12] were reviewed to identify (young) patients (age ⱕ 35 years) with a confirmed diagnosis of diffuse gastric cancer. Seven patients exhibited a familial clustering and met after pedigree analysis and pathologic review of the primary tumor the International Gastric Cancer Linkage Consortium (IGCLC) criteria for HDGC (ⱖ2 first-degree or second-degree relatives with diffuse gastric carcinoma, one of whom was diagnosed before age 50 years; or ⱖ3 cases of diffuse gastric carcinoma in first-degree or second-degree relatives, irrespective of age). All the patients were unrelated and came from Central Italy (Lazio, three patients) and Southern Italy (Puglia and Calabria, one and three patients, respectively). Written informed consent was obtained by the probands and their family members. 51 DNA Extraction For each patient DNA was extracted from paraffin-embedded tumor specimens as well as from normal gastric tissue or if still possible from peripheral blood mononuclear cells (PBMC). This latter source was utilized for relatives of patients. For paraffinembedded sections, paraffin was removed in serial passages in xylene and then the obtained pellet was resuspended in absolute ethanol. After treatment overnight at 50°C with proteinase K (Sigma, St. Louis MO; final concentration, 400 ␮g/ml), DNA was extracted using phenol– chlorophorm–isoamylic alcohol (25:24:1 v/v). PCR and DNA Sequence Analysis Primers utilized for amplification of each of the 16 exons of CDH1 were taken from previous studies [13]. Primers were synthetized by Pharmacia (Uppsala, Sweden). PCR reactions were performed using AmpliTaq (Applied Biosystems, Foster City, CA, USA) or AmpliTaq gold (Applied Biosystems; only for exons 3 and 13). The efficiency of PCR reactions was checked in a 2% agarose gel electrophoresis. DNA sequence analysis was performed from PCR reactions using an automated DNA sequencer (ABI Prism 310, Applied Byosistems) and the Big Dye terminator v3.1 staining kit (Applied Biosystems) according to the manufacturer’s instructions. Results were analyzed using the Seqscape v2.0 software package (Applied Biosystems). Reference sequence was EMBL Z13009. Methylation Promoter Study Promoter methylation is one of the most prominent mechanisms of gene silencing. The methylation status of the CDH1 promoter was performed using methylation-specific PCR (MSP) according to the protocol and primer design previously published. CDH1 promoter methylation analysis was performed using methylation-specific PCR according to the method described by Herman et al. [14] and using primers described by Hiraguri et al. for CpG island 3 of the CDH1 promoter [15]. Briefly, this assay entails the initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and the subsequent amplification with primers specific for methylated versus unmethylated DNA. Immunohistochemical Analysis (IHC) of CDH1 Tumor tissue biopsies were obtained at surgery. Tissue specimens were fixed in formalin and paraffin-embedded according to standard procedures. Four-micron sections of representative blocks from each case were deparaffinized in xylene. To identify the CDH1 protein expression, the Envision-peroxidase system (Dako, Glostrup, Denmark) was used. Clone G10 (Santa Cruz Biotechnology, Santa Cruz CA, USA) anti-human CDH1 primary antibody (1:20) in 1% bovine serum albumin–phosphate-buffered saline was used. Negative control for every experiment was done by replacing the primary antibody with albumin–phosphate-buffered saline. RESULTS All the HDGC patients underwent IHC of CDH1. A representative image is shown in Fig. 1. Results have shown that the protein was consistently downregulated in all patients. Faint protein levels were detectable only in the tumor area where the cancer cells maintained a minimal degree of cell-to-cell contact and a normal tissue architecture. For two patients having young susceptible relatives, gastroscopic analysis was performed and CDH1 expression was evalu- 52 JOURNAL OF SURGICAL RESEARCH: VOL. 121, NO. 1, SEPTEMBER 2004 FIG. 1. Representative IHC analysis from a healthy patient (A) and from HDGC (patient 7, panel B, C, and D). In A, normal gastric tissue displays pronounced membranous staining (magnification, ⫻100). HDGC results in loss of CDH1 staining. The arrows show typical ring cells of diffuse gastric cancer without expression of CDH1 (B, magnification, ⫻100; C and D, magnification, ⫻400). (Color version of figure is available online.) ated. CDH1 expression in all three relatives did not show alterations with respect to the CDH1 expression noticeable in healthy unrelated subjects (data not shown). Pedigrees for these two families is reported in Fig. 2. In all probands the CDH1 gene was sequenced in all its 16 exons and a summary of all the results is reported in Table 1. For each patient DNA genomic was extracted from cancer and PBMC. If PBMC were unavailable, DNA was extracted from a fragment of normal gastric tissue histologically checked for the absence of tumor foci. Six missense mutations were detected in the cancer tissue in five different patients. Representative electropherograms are shown in Fig. 3, while in Fig. 4 a site map for each mutation in the context of CDH1 gene is depicted. In two patients two mutations were found in exon 9: substitution C f T at nucleotide 1208 yielded the mutation A423V (patient 1), while substitution G f A at nucleotide 1222 determined the mutation A428T (patient 5). In two other patients mutations were found in exon 8: in patient 3 substitution G f A at nucleotide 1111 yielded the mutation B391N, whereas in patient 6 substitution A f C at nucleotide 1103 produced the mutation T388P. In the same patient an additional mutation was found in exon 16 at nucleotide 2510, where the transition G f C provoked the mutation G856R. Finally, patient 4 also exhibited a mutation in exon 16 at nucleotide 2530, where the transition A f G induced FIG. 2. Pedigrees of gastric cancer families for the families of patients 1 and 7. The squares represent male family members and the circles female family members; solid symbols indicate affected persons and open symbols unaffected persons. A slash over the symbol denotes death. An arrow identifies the individual screened for mutation in each family (proband). The age at diagnosis is indicated under each symbol. A double arrow indicates relatives for which CDH1 immunohistochemistry and CDH1 sequencing were performed. CONCOLINO ET AL.: HEREDITARY DIFFUSE GASTRIC CANCER TABLE 1 Summary of the Results Obtained through CDH1 IHC, DNA Sequencing, and Promoter Methylation Status in Cancer Specimens from HDCG Patients CDH1 IHC Patients analysis a 1 2 3 4 5 6 7 a b ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ Missense mutations b ⫹ (A423V) ⫺ ⫹ (B391N) ⫹ (S864G) ⫹ (A428T) ⫹ (T388P),(G856R) ⫺ Silent Promoter mutations b methylation ⫹ (A712A) ⫹ (A712A) ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ present; ⫺, not expressed or not present. Mutations were always present in heterozygosis. the mutation S864G. All six missense mutations were detected in heterozygosis. In addition to these six point mutations, one polymorphism was detected in two patients (1 and 2) in exon 13 at nucleotide 2076 transition C f T (A712A). Also such polymorphism was detected in heterozigosis. DNA sequence analysis performed in normal tissues revealed that all the patients did not carry DNA mutations in germ-line cells, since the mutations we found in cancer specimens were not detected in normal tissues. To confirm the absence of genetic mutations at CDH1 gene, the three relatives of patients 1 and 7 with diffuse gastric carcinoma were also sequenced for CDH1. As expected, no DNA mutations were detectable. To ascertain if DNA silencing by promoter methyl- 53 ation could be involved in CDH1 down-regulation, MSP was performed in all patients. Representative results are shown in Fig. 5. Four of five patients with DNA missense mutations also exhibited promoter methylation, whereas only patient 4 had a DNA missense mutation without promoter methylation. In patients 2 and 7, where DNA missense mutations were absent, promoter methylation also was not detectable despite the fact that also in this case CDH1 is downregulated in the tumor. DISCUSSION Diffuse gastric carcinoma is featured by a low CDH1 expression, dependent on genetic and epigenetic mechanisms [16]. In the case of genetic lesions, around 40% of patients display mutations in the central protein region coding for the extracellular domain of CDH1 and in particular for the Ca ⫹⫹ binding site [17]. It was previously demonstrated that such mutations are sufficient to disrupt CDH1 function, by hampering the correct protein folding and orientation [18]. Looking at the DNA mutations reported in our clinical settings of HDGC, mutations in exons 8 and 9 were found in the extracellular domain of CDH1. Both mutations at exon 8 (B391N and T388P) are sited in the Ca ⫹⫹ binding site of EC-3 domain, whereas mutations at exon 9 (A423V, A428T) are always in the EC-3 domain, but away from the Ca ⫹⫹ binding site. To our knowledge such mutations have not been previously reported and possible functional consequences resulting from such mutations are unknown. Two additional DNA missense mutations were noticed in exon 16. This sequence encodes FIG. 3. DNA missense mutations found in cancer specimens. In the upper panel a (patient 1), b (patient 3), c (patient 4), d (patient 5), e and f (patient 6) correspond to the wild-type sequence obtained from the correspondent normal tissue of each patient. In the lower panel DNA missense mutations noticed in cancer specimens (a⬘ A423V patient 1), b⬘ (B391N patient 3), c⬘ (S864G patient 4), d⬘ A428T patient 5, e⬘ and f⬘ (T388P and G856R, respectively, patient 6). 54 JOURNAL OF SURGICAL RESEARCH: VOL. 121, NO. 1, SEPTEMBER 2004 for the cytoplasmatic tail of the protein where interaction CDH1/␤-catenin occurs [19]. At this level there is a cluster of 8 serine, whose function is modulated by phosphorylation with the consequent regulation of CDH1 adhesivity [20]. One of these two mutations in exon 16 (S464G) was detected in one of the serine residues. The same mutation was also previously described by Berx et al. [21] and it is likely to affect the CDH-1-dependent signal transduction pathway. The additional mutation found at exon 16 (G856R) maps in the same CP-2 domain, interacting with catenins, but also such a mutation was never previously described. However, mutations found in cancer specimens were absent in germ-line cells from the same patients, thereby indicating that CDH-1 mutations occurred during the process of tumorigenesis. As to epigenetic alterations, we noticed gene silencing by promoter methylation in four cases. Anyway, in our setting all these patients exhibited both promoter methylation and DNA missense mutations, thereby prompting us to speculate that the two phenomena could be related and gene silencing could be driven by the missense mutations at CDH1. Since DNA mutations were always present in heterozygosis and promoter methylation is a reversible process, these facts suggest that novel therapeutic strategies able to reactivate the expression of silenced CDH1 normal allele could be helpful in the therapy of HDGC. However, in the absence of direct data proving a relationship, we cannot exclude that somatic CDH1 mutations and CDH1 down-regulation act through independent “private” pathways to induce functional inactivation and gene silencing, respectively, and that the concomitant combination of both these phenomena is casual in this clinical setting. Taking into consideration our findings, it appears that germ-line CDH1 cannot alone explain HDGC and that other genetic alterations are involved in this disease. In fact, somatic mutations, enhanced by gene silencing, underlie the CDH-1 down-regulation in five of seven patients. Remarkably, in two cases (patient 2 and patient 7) CDH1 was down-regulated in cancer specimens, without any evidence of either CDH1 mutations or gene silencing through promoter methylation. Therefore, other genetic unknown alterations FIG. 5. Representative MSP in cancer specimens of HDGC patients. Specific primers for the methylated (M) and unmethylated (U) promoter were used. The number at top is referred to as the patient code. Promoter methylation was found in patients 1, 3, 5, and 6. This experiment was repeated three times with identical results. able to induce CDH1 down-regulation could be present in the families affected by HDGC. This finding is well in keeping with recent data indicating a low prevalence of hereditary CDH1 genetic alterations in HDGC [22] and prompts further research in this field to isolate unknown genes involved in CDH1 down-regulation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. FIG. 4. Scheme reporting the mutations found in this clinical setting in the context of the CDH1 gene. Numbers are referred to as CDH1 exons. Sig (signal peptide), Precursor (protein precursor), Extracellular, TM (Transmembrane), and Cytoplasmic are the functional domains of CDH1. Each found mutation is inserted in the map as a bar. 11. Inberg, M., Lauren, P., and Viikari, S. 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