MRE11 expression is impaired in gastric cancer with microsatellite instability
Laura Ottini1,5,
Mario Falchetti1,2,
Calogero Saieva3,
Manola De Marco1,
Giovanna Masala3,
Ines Zanna3,
Milena Paglierani4,
Giuseppe Giannini1,
Alberto Gulino1,
Gabriella Nesi4,
Renato Mariani Costantini2 and
Domenico Palli3
1 Department of Experimental Medicine and Pathology, University La Sapienza, 00161 Rome, Italy, 2 Department of Oncology and Neurosciences, University Gabriele D'Annunzio and Center for the Study on Aging (Ce.S.I.), G. D'Annunzio Foundation, 66013 Chieti, Italy, 3 Molecular and Nutritional Epidemiology Unit, CSPO, Scientific Institute of Tuscany, 50100 Florence, Italy and 4 Department of Pathology, University of Florence, 50100 Florence, Italy
5 To whom correspondence should be addressed Email: ottini{at}yahoo.com
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Abstract
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Gastric carcinomas (GCs) with high-level microsatellite instability (MSI-H) are characterized by widespread mutations at coding and non-coding mononucleotide repeats. Deletions at coding mononucleotide tracts are predicted to cause frameshift mutations and alter normal protein functions. Mutations affecting non-coding mononucleotide repeats may lead to functional consequences if they occur in gene regulatory regions. To investigate whether mutations in non-coding polypyrimidine tracts within cancer-related genes may contribute to the phenotype of MSI-H GCs, we analysed the poly(T)11 tract constituting an accessory splicing signal within the intron 4 of the MRE11 gene. Mutations at the intronic MRE11 poly(T)11 were evaluated by PCR-based assay in 27 MSI-H, 22 MSI-low and 29 MSI-negative GCs derived from a well-characterized series of GCs identified in a high-risk area in Tuscany, Central Italy. Deletion of 2 and 1 bp at the MRE11poly(T)11 were identified in 33 and 48% MSI-H GCs, respectively. Biallelic mutations were frequently observed (77%) in GCs harbouring 2 bp deletions. The presence of MRE11poly(T)11 2 bp deletion was associated with a totally absent or strongly reduced MRE11 immunostaining (P < 0.001) and with a positive GC family history (P = 0.046). Immunoblotting assays confirmed the absence of MRE11 expression in GCs with a 2 bp deletion. The relatively high frequency of the MRE11poly(T)11 mutations, the occurrence of biallelic mutations and the evidence of loss of protein expression indicate MRE11 as novel mutational target in MSI-H GC. Overall, our results indicate that MSI-associated mutations occurring in non-coding repeats may affect protein expression in MSI-H GC.
Abbreviations: GC, gastric carcinoma; MMR, mismatch repair; MNR, hMRE11NBS1hRAD50 complex; MSI, microsatellite instability; MSI-H, high-level MSI; MSI-L, low-level MSI; MSS, MSI-negative
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Introduction
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Gastric carcinomas (GCs) with defective DNA mismatch repair (MMR) accumulate DNA replication errors at short sequence repeats. These tumours, estimated to comprise
1025% of all GCs (1,2), are characterized by widespread somatic mutations at coding and non-coding mononucleotide repeats and are identified by the presence of high-level microsatellite instability (MSI-H). Several lines of investigation indicate that MSI-H GCs follow a peculiar molecular pathway of tumour progression, characterized by the presence of frameshift mutations affecting coding mononucleotide tracts within cancer-related genes (3). In most cases, mutational events inactivate only one of the two alleles of target genes thus leading to haplo-insufficiency (46). Mutations in non-coding microsatellite sequences are frequently observed in MSI-H GCs but are unlikely to favour neoplastic growth unless they occur in gene regulatory regions that may control levels of gene expression. Non-coding polypyrimidine tracts at splicing acceptor sites are required for efficient spliceosome assembly and modulating branch site selection to splice mRNA correctly (7). In particular, polypyrimidine tracts with 11 continuous uridines are highly competitive pyrimidine tracts in splicing competition assays (8). Polypyrimidine repeat mutations may disrupt the correct mRNA splicing (9) but evidence that insertion/deletion occurring at these non-coding mononucleotide sequences may affect normal protein function has not yet been provided in MSI-related GC. We reported recently that mutations at the poly(T)11, constituting an accessory splicing signal within the 3'-splice acceptor site of MRE11 intron 4 (IVS-4), led to skipping of exon 5 and to the introduction of a premature stop codon in MMR-defective colon cancer cell lines (10). MRE11 is a multifunctional protein involved in double-strand break (DSB) repair and in signaling of the DNA damage response (11). Defects in MRE11 functions are associated with an ataxia-telangiectasia (AT)-like disorder, a cancer predisposition syndrome (12) and MRE11 mutations and aberrant transcript, resulting from abnormal splicing event, have been reported in primary tumours (13).
It is now well established that genomic instability is a hallmark of cancer and that mutations in genes involved in DNA damage response and repair increase the risk of tumour development. Interestingly, we have shown previously that mutations at the MRE11poly(T)11 are associated with a reduced expression of the three members of the hMRE11NBS1hRAD50 (MNR) complex, thus suggesting that the MNR complex function is impaired in MRE11 mutated cells (10). Moreover, the MMR proteins hMSH2, hMSH6 and hMLH1 reside together with ATM, BLM, BRCA1 and MNR complex within the BRCA1-associated genome surveillance (BASC) super-complex suggesting a link connecting MMR, DSBs and cell cycle checkpoint control (14).
To investigate whether MRE11 represent a mutational target in MSI-H GC and whether mutations in non-coding polypyrimidine tracts, acting as splicing acceptor signals in introns, may contribute to the progression of MSI-related GCs, a group of 27 MSI-H, 22 MSI-low (MSI-L) and 29 MSI-negative (MSS) GCs were identified from a well-characterized Italian high-risk population and analysed for mutations at the poly(T)11 within the branch-acceptor site in MRE11 IVS-4. MRE11 protein expression was investigated by immunohistochemistry and immunoblotting analyses in GCs harbouring either wild-type and mutated MRE11poly(T)11 alleles. Associations between clinico-pathological characteristics and MRE11 status were also investigated. Finally, to compare the frequency and to evaluate the association of MRE11 mutations with alterations of other genes involved in the control of genome stability, all MSI-H GCs were analysed for mutations in known target genes involved in the pathway of DNA damage response and repair.
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Materials and methods
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Study population
The current series of GC cases were identified in a high-risk area surrounding Florence, Central Italy. Patients were recruited in two different periods (19851987 and 19951996), while admitted in the Surgery Departments of the main hospitals of the area. In previous studies a sample of 126 of the original 382 GC cases of a population-based case-control study was tested for MSI (15,16). To expand this group, an additional series of 37 GC cases were identified in the frame of an epidemiological project on GC carried out in the same GC high-risk area (17). The histological specimens of these cases were retrieved but four cases were excluded because available tissue was insufficient for molecular analyses. Overall, MSI status was investigated in 159 paraffin-embedded specimens. An additional series of 18 fresh-frozen matched tumour and non-tumour tissue samples were obtained from a consecutive series of patients with GC, in the frame of an ongoing prospective study. All GC patients were residing in this high-risk area, signed an informed consent form and provided information on individual characteristics during a face-to-face interview, including a short section on family history of GC.
Pathological review
All GCs were confirmed histologically. Formalin-fixed, paraffin-embedded tumour blocks from the cases were retrieved from the archival files of the Pathology Department, Florence University, and all cases were reclassified, on the basis of the available slides, according to the Lauren, Ming and JRSGC classification for histological type and the WHO classification for grade of tumour differentiation (18,19).
MSI status and target gene mutation analyses
Step sections were cut from one representative paraffin-embedded block for each case; for each series of 10 consecutive sections, one was stained with haematoxylineosin and served as a guide for the microdissection of tumour and of normal tissue. Microdissection was performed using a sterile scalpel blade with the aid of a binocular microscope. DNA was extracted from matched tumour and normal tissue samples using standard procedures (15). A panel of six dinucleotide and two mononucleotide markers were used to determine the MSI status (15,16,20). Tumours were classified as MSI-H when showing contraction(s) in BAT26 and/or BAT25, as MSI-L when instability was limited to dinucleotide loci, and as MSS when no instability was observed at the loci tested (1,21). Mutations at coding repeats within seven genes involved in DNA repair [hMSH6 poly(C)8, hMSH3 poly(A)8, MED1 poly(A)10, RAD50 poly(A)9, BLM poly(A)9, ATR poly(A)10, BRCA2 poly(A)8] were analysed by a PCR-based assay using primer sets reported previously (20,22,23). PCRs, electrophoretic separation and autoradiography were as described previously (20). PCR results were always confirmed using three independent DNA extracts. PCR products were sequenced directly using the Sequenase PCR Sequencing Kit (USB), or alternatively, after insertion into a plasmid vector with the Topo TA Cloning Kit (Invitrogen).
MRE11 mutation analysis
A 122-bp fragment of MRE11 IVS-4, encompassing the poly(T)11 located at the 3'-splice site, was amplified with specifically designed primers (forward 5'-AATATTTTGGAGGAGAATCT-3'; reverse 5'-AATTGAAATGTTGAGGTTGCC-3'). PCR reactions, electrophoretic separation and autoradiography were as described previously (20). PCR products were sequenced directly by an ABI PRISM 377 DNA Sequencer (PE-Applied Biosystems). Two colon cancer cell lines, the WIDR [harbouring wild-type MRE11poly(T)11 alleles] and the MIP [harbouring MRE11poly(T)10/9 alleles] were used as controls for MRE11 poly(T)11 mutations. Results were always confirmed using three independent DNA extracts.
Immunohistochemistry
Immunohistochemical assay for MRE11 protein was performed in all 27 GCs with the MSI-H phenotype. Immunoperoxidase staining was performed on 5-µm-thick sections. After dewaxing and blocking with endogenous peroxidase, sections were pretreated by microwave for 25 min (MicroMed T/T Mega, Milestone, BG, Italy), washed and incubated with mouse anti-MRE11 monoclonal antibody (Novus Biologicals, Littleton, CO) diluted 1:200 for 60 min at room temperature. Antigen-bound primary antibody was detected using a standard streptavidinbiotin peroxidase method (Lab Vision, Fremont, CA). Immunostaining was visualized with diaminobenzidine-hydrogen peroxidase (BioGenex, San Ramon, CA). Sections were lightly counterstained with haematoxylin. Tumours were scored in a semi-quantitative method according to the following scheme: no expression (), no signal in any tumour cell; severe reduction (/+), positive signal in <50% of tumour cells; normal expression (+), positive signal in >50% of tumour cells. For immunoreactivity of anti-MRE11 antibody only nuclear staining was acceptable; cytoplasmic positivity was infrequently encountered and was considered artifactual. Nuclear staining intensity was noted but was not used in determination of the final reactivity score. In each case, normal tissue adjacent to the tumour was used as an internal control.
Lysate preparation and immunoblotting
Matched tumour and normal gastric mucosa, from fresh-frozen GCs, were homogenated in lysis buffer (10 mM Tris, pH 7.5/100 mM NaCl/5 mM EDTA/0.5% NP-40) supplemented with protease and phosphatase inhibitors. Typically, we used 40 µg of protein for direct immunoblot analyses. Lysates were resolved by electrophoresis on 9% SDSpolyacrylamide gels and transferred O/N to nitrocellulose. Membranes were blocked for 1 h at room temperature in TBS-T (10 mM Tris, pH 8/150 mM NaCl/0.1% Tween 20) with 5% dry milk and then incubated for 1 h at room temperature with anti-MRE11 polyclonal antibody (Abcam Limited, Cambridge, Cambridgeshire, UK) diluted 1:8000 and anti-PCNA monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), used as a control for nuclear proteins, diluted 1:5000. Signals were developed by enhanced chemiluminescence reaction (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK).
Statistical analysis
The database with extensive individual information was merged with the histological data and laboratory assay results. The resulting dataset was analysed by the statistical package SAS. Analyses were carried out according to a classification of MRE11 status in two major categories: GCs with a 2 bp deletion and GCs with a 1 bp deletion/wild-type alleles. Comparisons for different variables were performed using the two-tailed Fisher exact test (for two levels) and the
2 test for trend or the
2 test (for more than two levels), as appropriate. A non-parametric test (MannWhitney test) was used to compare the median values of mutations in the two major categories of cases. A P value <0.05 was considered significant.
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Results
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The MRE11 poly(T)11 is a mutational target in MSI-H gastric cancer
To investigate whether MRE11 may represent a mutational target in GC with the MSI phenotype, MSI status was evaluated in a series of 159 GC cases. Overall, 110 tumours (69%) were classified as MSS, 22 (14%) MSI-L and 27 (17%) MSI-H. The poly(T)11 within the 3'-splice acceptor site of MRE11 IVS-4 was analysed in 27 MSI-H, 22 MSI-L and 29 MSS GCs. Abnormal PCR products were observed in tumour DNA of 22/27 (81%) MSI-H GCs. No alterations were observed in the MSI-L and MSS GCs analysed. Mutations were all represented by contractions, in particular, 48 (13/27) and 33% (9/27) MSI-H GCs harboured 1 and 2 bp deletions, respectively (Table I). Interestingly, biallelic mutations (i.e. 1/2 bp, 2/2 bp) resulting in MRE11poly(T)9/(T)10 and poly(T)9/(T)9 alleles, were observed frequently in GCs with 2 bp deletions (7/9, 77%). The individual characteristics of each GC case and MRE11 status are reported in Table II. Table III shows the associations between MRE11 status and clinico-pathological characteristics. Analyses were carried out according to a classification of MRE11 status in two categories: GCs with a 2 bp deletion and GCs with a 1 bp deletion/wild-type alleles. A borderline positive association between 2 bp deletions and GC family history in first-degree relatives was found (P = 0.046): 7/9 (78%) of the GCs with a 2 bp deletion reported positive GC family history versus 6/18 (33%) of the GCs with a 1 bp deletion/wild-type alleles. Interestingly, 5/7 cases with positive GC family history reported the occurrence of GC in their mother, including one case that also reported GC in his father and in one sibling (#19), and one case who also reported colon cancer in a sister (#25). A tendency to an association with vascular invasion (P = 0.071) also emerged: histological evidence of vascular invasion was found in all the nine GCs with a 2 bp deletion, but only in 67% of the GCs with a 1 bp deletion/wild-type alleles. No associations were found with other clinico-pathologic characteristics and overall survival at 10 years.
Deletions at the MRE11 poly(T)11 impair MRE11 protein expression in gastric cancer
To verify whether deletions at the poly(T)11 in MRE11 IVS-4 might affect MRE11 protein expression, immunohistochemical analysis was performed in the 27 MSI-H GCs. Two observers (G.N. and M.P.) assessed jointly the immunostaining without prior knowledge of the molecular data. Overall, 15/27 (56%) tumours showed normal expression, 6/27 (22%) severe reduction and 6/27 (22%) no expression of MRE11 (Table IV). A statistically significant association (P < 0.001) emerged between MRE11poly(T)11 2 bp deletion and loss or strongly reduced MRE11 protein expression. The majority of the cases harbouring wild-type MRE11poly(T)11 or a 1 bp deletion (15/18) showed normal MRE11 expression (Table IV). In contrast, all GCs cases with a 2 bp deletion showed an absence or severe reduction of MRE11 expression. In particular, of the nine GCs with a 2 bp deletion, five cases showed a total absence of MRE11 immunostaining and four cases showed a significant decrease in MRE11 expression. Within the four GCs showing a significant decrease in MRE11 expression, areas characterized by normal, slightly reduced and absent MRE11 immunostaining were observed and microdissected (Figure 1A). Mutational analysis (Figure 1B) and direct sequencing (Figure 1C), performed on DNA extracted from each distinct area, revealed the presence of an MRE11poly(T)11 2 bp deletion in tumour DNA derived from areas with an absence of MRE11 expression and, interestingly, of a 1 bp deletion (not detectable when the entire tumour was analysed) in tumour DNA obtained from areas with slightly reduced MRE11 expression. These results suggest that the degree of MRE11poly(T)11 deletion might differently affect the expression of the MRE11 protein in GC. To further investigate MRE11 expression in vivo, we performed western blotting analyses on 18 GCs, for which frozen samples were available. These GCs were first characterized for the MSI status; two cases resulted in MSI-H and harboured the MRE11 mutation. In particular, one case (#GC13f) had an heterozygous 1 bp deletion resulting in poly(T)10/(T)11 alleles, and the other (#GC9f) showed a 2 bp deletion resulting in poly(T)9/(T)10 alleles (Figure 2A). Immunoblotting analysis was performed on lysates of paired normal gastric mucosa and tumours obtained from these two GCs and from a case (#GC12f) with wild-type MRE11poly(T)11. Wild-type MRE11 protein expression was observed in the normal gastric mucosa of all GCs analysed and in the WIDR colon cancer cell line, used as positive control (Figure 2B). In contrast, MRE11 protein expression was evident only in a tumour from the case with MRE11 wild-type (#GC12f) and poly(T)10/(T)11 alleles (GC13f), no MRE11 expression was observed in the case (#GC9f) bearing poly(T)9/(T)10 alleles (Figure 2B). Taken together, these findings demonstrated that the degree of MRE11poly(T)11 deletions differently affect the expression of MRE11 in GC, with large deletions (i.e. 2 bp) causing loss or severe reduction of expression.
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Table IV. Distribution of the 27 MSI-H GCs according to MRE11 immunohistochemical expression and mutational status
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Fig. 1. Immunohistochemical and mutational analyses of MRE11 in a GC sample with severe reduction of MRE11 expression (GC case #21). (A) Gastric mucosa (M) adjacent to the tumour demonstrating intense positive nuclear MRE11 staining. Tumour areas showing severe reduction (T1) and complete loss (T2) of MRE11 staining (original magnification 20x). (B) MRE11poly(T)11 mutational analysis performed on DNA obtained from microdissected areas (M, T1, T2) of GC case #21. M, DNA from gastric mucosa expressing MRE11; T1, DNA from tumour areas with reduced MRE11 expression; T2, DNA from tumour areas with loss of MRE11 expression. Arrows indicate alleles with deletion of 1 (1bp del) and 2 bp (2bp del). T1 showed a 1 bp deletion and T2 showed both 1 and 2 bp deletion. (C) Electropherograms showing the profiles of MRE11 wild-type poly(T)11 in M, the coexistence of poly(T)10/(T)11 alleles in T1 and the coexistence of poly(T)9/(T)10 alleles in T2.
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Fig. 2. Mutational analysis and immunoblotting of MRE11 in GCs. (A) MRE11poly(T)11 mutational analysis performed on DNA obtained from frozen samples of GCs (cases #GC12f, GC9f, GC13f). N, DNA from normal gastric mucosa; T, tumour DNA. Arrows indicate alleles with deletion of 1 (1 bp del) and 2 bp (2 bp del). Case #GC12f resulted with MRE11 wild-type alleles, case #GC9f with 1 and 2 bp deletion and case #GC13f with a 1 bp deletion. (B) Western blotting analyses of MRE11 in GC samples with different MRE11poly(T) mutations (cases #GC12f, GC9f, GC13f). Immunoblotting analysis was performed on lysates obtained from paired normal mucosa (N) and tumour (T) of GCs and from the WIDR colon cancer cell line, used as positive control. Wild-type MRE11 protein expression was observed in normal gastric mucosa of all the GCs analysed and in the WIDR colon cancer cell line. In contrast, MRE11 protein expression was evident only in the tumour of cases #GC12f and GC13f, respectively, harbouring MRE11 wild-type and poly(T)10/(T)11 alleles, no MRE11 expression was observed in case #GC9f that showed poly(T)9/(T)10 alleles. PCNA expression was used to normalize the results.
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Frameshift mutations in target genes involved in surveillance of DNA stability
To evaluate the frequency of MRE11 mutations in comparison with other known target genes and to verify possible associations between the MRE11 status and frameshift mutations in genes involved in DNA damage response, all 27 MSI-H GCs were analysed for somatic mutations at coding repeats within genes involved in MMR (hMSH6, hMSH3, MED1) and DSBs (RAD50, BLM, BRCA2, ATR). Mutations were identified in 24/27 (88%) MSI-H GCs and consisted mainly in 1 bp deletion (44/45, 98%). A biallelic mutation, consisting of a 1 bp deletion and a 1 bp insertion, was observed in only one case (1/45, 2%) at the hMSH6poly(C)8. In the other cases, the occurrence of a homozygous 1 bp deletion is unlikely because the wild-type allele signal was always present, with a reduction in intensity within the 50% range (data not shown). With regard to mutation frequency, the pathway of MMR hMSH6 was altered in 41% (11/27), hMSH3 in 37% (10/27) and MED1 in 19% (5/27) of the MSI-H GCs. Considering the DSBs repair pathway ATR was mutated in 33% (9/27), BLM in 18% (5/27) and RAD50 in 18% (5/27) of the MSI-H GCs. No significant association emerged between the MRE11 status and the target genes analysed (Table V). On average, each case in the group with a 2 bp deletion showed 2.0 mutations in the target genes analysed, in comparison with a mean value of 1.5 mutations among cases with a 1 bp deletion/wild-type alleles (P = 0.321).
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Table V. Distribution of 27 MSI-H GC cases according to the presence of mutations in the seven DNA repair genes analysed and MRE11 status; cumulative and average number of mutations
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Discussion
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In this paper, we provide evidence that MSI-associated mutations occurring in non-coding sequences controlling the levels of gene expression may represent a target of selection in MSI-H GCs. It is well established that MSI-H GC have an increased tendency to accumulate DNA replication errors at coding and non-coding repetitive tracts (13,6). The functional relevance of mutations at coding mononucleotide repeats is predicted by the inactivation of target genes due to frameshift mutations. In contrast, a functional consequence of mutations at non-coding repetitive sequences may be assumed if they occur in gene regulatory regions that may affect normal protein functions (6). In this regard, we reported recently that deletions at the poly(T)11 located within an accessory splicing signal in the IVS-4 of the MRE11 gene lead to the skipping of exon 5 and to the introduction of a premature stop codon (10). Interestingly, these mutations were selectively found in MMR-defective colon cancer cell lines suggesting that MRE11 might identify a novel target in MSI-associated cancer. In the present study, we showed that MRE11 represents a mutational target in MSI-related GC. In fact, MRE11poly(T)11 mutations were observed in 81% of the MSI-H GCs and, in particular, 1 and 2 bp deletions occurred in 48 and 33% MSI-H GCs, respectively. In contrast, the MSI-L and MSS GCs analysed do not show MRE11poly(T)11 mutations. Mutation frequency of coding repeats within genes potentially involved in human carcinogenesis has been proposed as one of the criteria to distinguish real targets (i.e. genes mutated because of MMR-deficiency and playing a role in neoplastic growth) from bystanders (i.e. genes that happen to be mutated by chance in a cell that subsequently develops clonal growth advantage) (24,25). In this respect, the frequency of MRE11 functional mutations (i.e. 2 bp deletion) here reported in MSI-H GCs is quite comparable with values obtained for real targets, and the relatively high incidence of MRE11 mutations is suggestive of a positive pressure towards the selection in MSI-H GCs. In addition to mutation frequency, genetic (i.e. biallelic mutations) and functional (i.e. loss of function) evidence is required to identify real target genes. Interestingly, we observed that 77% of the GCs with MRE11 larger mutations (i.e. 2 bp deletion) harboured biallelic mutations, thus suggesting impairment of the splicing of MRE11 precursor transcript from both alleles. In addition, we showed that MSI-H GCs with biallelic/2 bp deletions manifested loss of wild-type MRE11 protein expression, thus indicating loss of MRE11 function. Moreover, here we found a statistically significant association (P = 0.0002) between MRE11poly(T)11 2 bp deletion and loss of MRE11 protein expression, as detected by immunohistochemistry. Recently, a correlation in the level of expression of the three components of the MNR (MRE11, NBS1 and RAD50) detected by immunohistochemistry in breast carcinomas has been found, although in this type of tumour the mechanism that leads to the deregulation of the expression of these proteins is not known (26). We showed previously that, in MMR-defective colon cancer cell lines, deletions at the MRE11poly(T)11 are associated with a reduced production of wild-type MRE11 transcript and protein and that impairment of MRE11 protein expression correlated with reduced expression of NBS1 and RAD50, thus indicating that MRE11 is required for the correct assembly and stability of all the components of the MNR complex (10). Taken together, all these findings suggest that the reduced expression of MRE11 is likely to result in the functional impairment of the MNR complex in MMR-deficient GCs. This is of particular interest in the context of gastric carcinogenesis considering that the MNR complex plays a relevant role in protecting cells from deleterious DNA-DSBs that might be generated by endogenous and exogenous mutagens. Intriguingly, environmental and dietary carcinogen exposures are known to be implicated in the process of gastric carcinogenesis and suspected risk factors for GC include chemical carcinogens, present in cigarette smoke and some food items (i.e. polycyclic aromatic hydrocarbons), that produce bulky DNA adducts and DNA-DSBs. It is now well established that mutations in genes involved in DNA damage repair increase the risk of tumour development and environmental exposures may magnify genetic defects. Indeed, genes involved in the surveillance of DNA stability are targets for mutations in MSI-related gastrointestinal cancer. In the present study, 88% of MSI-H GCs showed to accumulate heterozygous somatic mutations in multiple genes encoding components of the MNR, MMR and DSBs complexes. Considering the link connecting MNR, MMR and DSBs via the BASC super-complex, and according to the haplo-insufficiency model (46,22), accumulation of mutations in multiple genes whose products have synergistic roles at different points in a specific pathway may lead to impairment of the DNA repair pathways in which the super-complex is thought to operate.
Looking at the possible correlations between MRE11 mutations and clinical-pathological characteristics we found a borderline positive association between 2 bp deletions and GC family history (P = 0.046). In particular, our results suggested a specific association with a positive family history for GC in the mother. Interestingly, in a previous paper, based on a large Italian case-control study, we reported that GC risk was specifically increased for a positive family history in the mother but not when the affected parent was the father (27). The significance of this finding needs to be elucidated. Intriguingly, a higher frequency of MRE11 mutations was also observed among familial compared with sporadic colon cancer cases (28).
In conclusion, this study showed that mutations of the intronic poly(T)11 repeat, within the branch-acceptor site in MRE11, are selectively associated with the MSI-H phenotype in GC and with impairment of MRE11 expression. The relative high frequency of MRE11poly(T)11 deletions, the occurrence of biallelic mutations and the evidence of loss of protein expression indicate MRE11 as a novel mutational target in MSI-H GCs. Other mechanisms affecting MRE11 expression may be taken into account: the occurrence of alternative mutations in MRE11 or in other gene regulatory regions, the presence of mutations in other genes coding for the MNR complex and the possibility of an imprinting effect on one of the MRE11 alleles. Overall, these results represent the first evidence that mutations at non-coding mononucleotide repeats within gene regulatory regions may lead to functional consequences in MSI-H GCs and suggest that the functional significance of mutations in other intronic repeats needs to be investigated.
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Acknowledgments
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The authors wish to thank all the patients participating into the study for their collaboration, Drs Roberto Manetti and Renato Moretti (Surgery Department, A.O. Careggi, Firenze) for help with obtaining frozen tissue samples and Francesco Sera (CSPO) for helpful comments. The study was supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC) to L.O. and R.M.C.; from Consiglio Nazionale delle Ricerche (CNR, Strategic Project Oncology 2003) to R.M.C.; from Istituto Superiore di Sanità (ISS) to D.P.. M.F. is a fellow of the Fondazione Italiana Ricerca sul Cancro (FIRC).
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Received May 11, 2004;
revised July 17, 2004;
accepted August 2, 2004.