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Vuković Đerfi K, Salar A, Cacev T, Kapitanović S. EMAST Type of Microsatellite Instability-A Distinct Entity or Blurred Overlap between Stable and MSI Tumors. Genes (Basel) 2023; 14:1474. [PMID: 37510378 PMCID: PMC10380056 DOI: 10.3390/genes14071474] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2023] [Revised: 07/15/2023] [Accepted: 07/18/2023] [Indexed: 07/30/2023] Open
Abstract
Microsatellite instability (MSI) represents an accumulation of frameshifts in short tandem repeats, microsatellites, across the genome due to defective DNA mismatch repair (dMMR). MSI has been associated with distinct clinical, histological, and molecular features of tumors and has proven its prognostic and therapeutic value in different types of cancer. Recently, another type of microsatellite instability named elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) has been reported across many different tumors. EMAST tumors have been associated with chronic inflammation, higher tumor stage, and poor prognosis. Nevertheless, the clinical significance of EMAST and its relation to MSI remains unclear. It has been proposed that EMAST arises as a result of isolated MSH3 dysfunction or as a secondary event in MSI tumors. Even though previous studies have associated EMAST with MSI-low phenotype in tumors, recent studies show a certain degree of overlap between EMAST and MSI-high tumors. However, even in stable tumors, (MSS) frameshifts in microsatellites can be detected as a purely stochastic event, raising the question of whether EMAST truly represents a distinct type of microsatellite instability. Moreover, a significant fraction of patients with MSI tumors do not respond to immunotherapy and it can be speculated that in these tumors, EMAST might act as a modifying factor.
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Affiliation(s)
- Kristina Vuković Đerfi
- Laboratory for Personalized Medicine, Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka cesta 54, 10000 Zagreb, Croatia
| | - Anamarija Salar
- Laboratory for Personalized Medicine, Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka cesta 54, 10000 Zagreb, Croatia
| | - Tamara Cacev
- Laboratory for Personalized Medicine, Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka cesta 54, 10000 Zagreb, Croatia
| | - Sanja Kapitanović
- Laboratory for Personalized Medicine, Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka cesta 54, 10000 Zagreb, Croatia
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Raeker MO, Carethers JM. Immunological Features with DNA Microsatellite Alterations in Patients with Colorectal Cancer. JOURNAL OF CANCER IMMUNOLOGY 2020; 2:116-127. [PMID: 33000102 DOI: 10.33696/cancerimmunol.2.024] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Competent human DNA mismatch repair (MMR) corrects DNA polymerase mistakes made during cell replication to maintain complete DNA fidelity in daughter cells; faulty DNA MMR occurs in the setting of inflammation and neoplasia, creating base substitutions (e.g. point mutations) and frameshift mutations at DNA microsatellite sequences in progeny cells. Frameshift mutations at DNA microsatellite sequences are a detected biomarker termed microsatellite instability (MSI) for human disease, as this marker can prognosticate and determine therapeutic approaches for patients with cancer. There are two types of MSI: MSI-High (MSI-H), defined by frameshifts at mono- and di-nucleotide microsatellite sequences, and elevated microsatellite alterations at selected tetranucleotide repeats or EMAST, defined by frameshifts in di- and tetranucleotide microsatellite sequences but not mononucleotide sequences. Patients with colorectal cancers (CRCs) manifesting MSI-H demonstrate improved survival over patients without an MSI-H tumor, driven by the generation of immunogenic neoantigens caused by novel truncated proteins from genes whose sequences contain coding microsatellites; these patients' tumors contain hundreds of somatic mutations, and show responsiveness to treatment with immune checkpoint inhibitors. Patients with CRCs manifesting EMAST demonstrate poor survival over patients without an EMAST tumor, and may be driven by a more dominant defect in double strand break repair attributed to the MMR protein MSH3 over its frameshift correcting function; these patients' tumors often have a component of inflammation (and are also termed inflammation-associated microsatellite alterations) and show less somatic mutations and lack coding mononucleotide frameshift mutations that seem to generate the neoantigens seen in the majority of MSI-H tumors. Overall, both types of MSI are biomarkers that can prognosticate patients with CRC, can be tested for simultaneously in marker panels, and informs the approach to specific therapy including immunotherapy for their cancers.
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Affiliation(s)
- Maide O Raeker
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan
| | - John M Carethers
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan.,Department of Human Genetics and Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan
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Kasela M, Nyström M, Kansikas M. PMS2 expression decrease causes severe problems in mismatch repair. Hum Mutat 2019; 40:904-907. [PMID: 30946512 PMCID: PMC6618857 DOI: 10.1002/humu.23756] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2018] [Revised: 03/15/2019] [Accepted: 03/24/2019] [Indexed: 12/21/2022]
Abstract
PMS2 is one of the four susceptibility genes in Lynch syndrome (LS), the most common cancer syndrome in the world. Inherited mutations in DNA mismatch repair (MMR) genes, MLH1, MSH2, and MSH6, account for approximately 90% of LS, while a relatively small number of LS families segregate a PMS2 mutation. This and the low cancer penetrance in PMS2 families suggest that PMS2 is only a moderate or low‐risk susceptibility gene. We have previously shown that even a partial expression decrease in MLH1, MSH2, or MSH6 suggests that heterozygous LS mutation carriers have MMR malfunction in constitutive tissues. Whether and how PMS2 expression decrease affects the repair capability is not known. Here, we show that PMS2 knockdown cells retaining 19%, 33%, or 53% of PMS2 expression all have significantly reduced MMR efficiency. Surprisingly, the cells retaining expression levels comparable to PMS2 mutation carriers indicate the lowest repair efficiency.
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Affiliation(s)
- Mariann Kasela
- Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland
| | - Minna Nyström
- Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland
| | - Minttu Kansikas
- Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland
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Abstract
Lynch Syndrome (LS) is the most common dominantly inherited colorectal cancer (CRC) predisposition and is caused by a heterozygous germline defect in one of the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, or PMS2. High microsatellite instability (MSI-H) and loss of MMR protein expression in tumours reflecting a defective MMR are indicators for LS, as well as a positive family history of early onset CRC. MSH2 and MSH6 form a major functional heterodimer, and MSH3 is an alternative binding partner for MSH2. So far, the role of germline MSH3 variants remains unclear, as to our knowledge heterozygous truncating variants are not regarded causative for LS, but were detected in patients with CRC, and recently biallelic MSH3 defects have been identified in two patients with adenomatous polyposis. By gene screening we investigated the role of MSH3 in 11 LS patients with truncating MSH6 germline variants and an unexplained MSH2 protein loss in their corresponding MSI-H tumours. We report the first two LS patients harbouring heterozygous germline variants c.1035del and c.2732T>G in MSH3 coincidentally with truncating variants in MSH6. In the patient with truncating germline variants in MSH3 and MSH6, two additional somatic second hits in both genes abrogate all binding partners for the MSH2 protein which might subsequently be degraded. The clinical relevance of MSH3 germline variants is currently under re-evaluation, and heterozygous MSH3 defects alone do not seem to induce a LS phenotype, but might aggravate the MSH6 phenotype in affected family members.
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Koi M, Tseng-Rogenski SS, Carethers JM. Inflammation-associated microsatellite alterations: Mechanisms and significance in the prognosis of patients with colorectal cancer. World J Gastrointest Oncol 2018; 10:1-14. [PMID: 29375743 PMCID: PMC5767788 DOI: 10.4251/wjgo.v10.i1.1] [Citation(s) in RCA: 38] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/26/2017] [Revised: 11/29/2017] [Accepted: 12/06/2017] [Indexed: 02/05/2023] Open
Abstract
Microsatellite alterations within genomic DNA frameshift as a result of defective DNA mismatch repair (MMR). About 15% of sporadic colorectal cancers (CRCs) manifest hypermethylation of the DNA MMR gene MLH1, resulting in mono- and di-nucleotide frameshifts to classify it as microsatellite instability-high (MSI-H) and hypermutated, and due to frameshifts at coding microsatellites generating neo-antigens, produce a robust protective immune response that can be enhanced with immune checkpoint blockade. More commonly, approximately 50% of sporadic non-MSI-H CRCs demonstrate frameshifts at di- and tetra-nucleotide microsatellites to classify it as MSI-low/elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) as a result of functional somatic inactivation of the DNA MMR protein MSH3 via a nuclear-to-cytosolic displacement. The trigger for MSH3 displacement appears to be inflammation and/or oxidative stress, and unlike MSI-H CRC patients, patients with MSI-L/EMAST CRCs show poor prognosis. These inflammatory-associated microsatellite alterations are a consequence of the local tumor microenvironment, and in theory, if the microenvironment is manipulated to lower inflammation, the microsatellite alterations and MSH3 dysfunction should be corrected. Here we describe the mechanisms and significance of inflammatory-associated microsatellite alterations, and propose three areas to deeply explore the consequences and prevention of inflammation's effect upon the DNA MMR system.
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Affiliation(s)
- Minoru Koi
- Division of Gastroenterology, Department of Internal Medicine and Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI 48109-5368, United States
| | - Stephanie S Tseng-Rogenski
- Division of Gastroenterology, Department of Internal Medicine and Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI 48109-5368, United States
| | - John M Carethers
- Division of Gastroenterology, Department of Internal Medicine and Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI 48109-5368, United States
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Itkonen HM, Kantelinen J, Vaara M, Parkkinen S, Schlott B, Grosse F, Nyström M, Syväoja JE, Pospiech H. Human DNA polymerase α interacts with mismatch repair proteins MSH2 and MSH6. FEBS Lett 2016; 590:4233-4241. [DOI: 10.1002/1873-3468.12475] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2016] [Revised: 10/20/2016] [Accepted: 10/26/2016] [Indexed: 12/11/2022]
Affiliation(s)
- Harri M. Itkonen
- Research group Biochemistry; Leibniz Institute on Aging - Fritz Lipmann Institute; Jena Germany
- Department of Biology; University of Eastern Finland; Joensuu Finland
- Prostate Cancer Research Group; Nordic EMBL Partnership; Centre for Molecular Medicine Norway; University of Oslo; Blindern Norway
| | - Jukka Kantelinen
- Department of Biosciences; Division of Genetics; University of Helsinki; Finland
| | - Markku Vaara
- Department of Biology; University of Eastern Finland; Joensuu Finland
| | - Sinikka Parkkinen
- Department of Biology; University of Eastern Finland; Joensuu Finland
| | - Bernhard Schlott
- Research group Biochemistry; Leibniz Institute on Aging - Fritz Lipmann Institute; Jena Germany
- Service group Proteomics; Leibniz Institute on Aging - Fritz Lipmann Institute; Jena Germany
| | - Frank Grosse
- Research group Biochemistry; Leibniz Institute on Aging - Fritz Lipmann Institute; Jena Germany
| | - Minna Nyström
- Department of Biosciences; Division of Genetics; University of Helsinki; Finland
| | - Juhani E. Syväoja
- Institute of Biomedicine; University of Eastern Finland; Kuopio Finland
| | - Helmut Pospiech
- Research group Biochemistry; Leibniz Institute on Aging - Fritz Lipmann Institute; Jena Germany
- Faculty of Biochemistry and Molecular Medicine; University of Oulu; Finland
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Inserting Extrahelical Structures into Long DNA Substrates for Single-Molecule Studies of DNA Mismatch Repair. Methods Enzymol 2016; 582:221-238. [PMID: 28062036 DOI: 10.1016/bs.mie.2016.08.006] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
The DNA mismatch repair (MMR) system corrects errors that occur during DNA replication. MMR needs the coordinated and highly dynamic assembly of repair enzymes at the site of the lesion. By visualizing transient intermediates of these assemblies, single-molecule approaches have shed critical insights into the mechanisms of MMR. These studies frequently require long (>20kb) DNA substrates with lesions and other extrahelical structures inserted at defined positions. DNA derived from bacteriophage λ (λ-DNA) is a high quality long (48.5kb) DNA substrate that is frequently used in single-molecule studies. Here we provide detailed protocols for site-specific incorporation of recombinant sequences and extrahelical structures into λ-DNA. We also describe how to assemble DNA curtains, and how to collect and analyze single-molecule observations of lesion recognition by MMR proteins diffusing on these DNA curtains. These protocols will facilitate future single-molecule studies of DNA transcription, replication, and repair.
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Kansikas M, Kasela M, Kantelinen J, Nyström M. Assessing how reduced expression levels of the mismatch repair genes MLH1, MSH2, and MSH6 affect repair efficiency. Hum Mutat 2014; 35:1123-7. [PMID: 24924810 DOI: 10.1002/humu.22605] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2014] [Accepted: 06/09/2014] [Indexed: 12/30/2022]
Abstract
Lynch syndrome (LS), the most common familial colon cancer, is associated with mismatch repair (MMR) malfunction. As mutation carriers inherit one normal and one defected MMR gene allele, cancer risk can be considered as limited amount of normal MMR gene product. How reductions in different MMR gene expressions affect MMR capability is, however, not known. The in vitro MMR assay is a method for the pathogenicity assessment of MMR gene variants causing functional or expressional defects and thus also suitable to evaluate the effects of reduced expression of normal mRNA. Here, the assay was applied to quantify repair efficiencies of human cells retaining varying expression levels (25%/50%/75%) of the main LS susceptibility genes MLH1, MSH2, or MSH6. Compared with the shRNA knockdown control, already a 50% reduction in mRNA levels could be detected as decreased MMR function although without statistical significance in MLH1. In MSH2 and MLH1, total loss of MMR was achieved with 25% expression, whereas in MSH6 and MSH2, the repair capability decreased significantly already with 75% expression. Our results provide a preliminary indication of relative expressions required for wild-type function and suggest that the in vitro MMR assay could be used to recognize expression levels indicative of LS.
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Affiliation(s)
- Minttu Kansikas
- Department of Biosciences, Division of Genetics, University of Helsinki, Helsinki, Finland
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9
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Functional analysis in mouse embryonic stem cells reveals wild-type activity for three MSH6 variants found in suspected Lynch syndrome patients. PLoS One 2013; 8:e74766. [PMID: 24040339 PMCID: PMC3769292 DOI: 10.1371/journal.pone.0074766] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2011] [Accepted: 08/06/2013] [Indexed: 02/02/2023] Open
Abstract
Lynch syndrome confers an increased risk to various types of cancer, in particular early onset colorectal and endometrial cancer. Mutations in mismatch repair (MMR) genes underlie Lynch syndrome, with the majority of mutations found in MLH1 and MSH2. Mutations in MSH6 have also been found but these do not always cause a clear cancer predisposition phenotype and MSH6-defective tumors often do not show the standard characteristics of MMR deficiency, such as microsatellite instability. In particular, the consequences of MSH6 missense mutations are challenging to predict, which further complicates genetic counseling. We have previously developed a method for functional characterization of MSH2 missense mutations of unknown significance. This method is based on endogenous gene modification in mouse embryonic stem cells using oligonucleotide-directed gene targeting, followed by a series of functional assays addressing the MMR functions. Here we have adapted this method for the characterization of MSH6 missense mutations. We recreated three MSH6 variants found in suspected Lynch syndrome families, MSH6-P1087R, MSH6-R1095H and MSH6-L1354Q, and found all three to behave like wild type MSH6. Thus, despite suspicion for pathogenicity from clinical observations, our approach indicates these variants are not disease causing. This has important implications for counseling of mutation carriers.
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10
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Delpu Y, Cordelier P, Cho WC, Torrisani J. DNA methylation and cancer diagnosis. Int J Mol Sci 2013; 14:15029-15058. [PMID: 23873296 PMCID: PMC3742286 DOI: 10.3390/ijms140715029] [Citation(s) in RCA: 120] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2013] [Revised: 06/28/2013] [Accepted: 07/04/2013] [Indexed: 02/06/2023] Open
Abstract
DNA methylation is a major epigenetic modification that is strongly involved in the physiological control of genome expression. DNA methylation patterns are largely modified in cancer cells and can therefore be used to distinguish cancer cells from normal tissues. This review describes the main technologies available for the detection and the discovery of aberrantly methylated DNA patterns. It also presents the different sources of biological samples suitable for DNA methylation studies. We discuss the interest and perspectives on the use of DNA methylation measurements for cancer diagnosis through examples of methylated genes commonly documented in the literature. The discussion leads to our consideration for why DNA methylation is not commonly used in clinical practice through an examination of the main requirements that constitute a reliable biomarker. Finally, we describe the main DNA methylation inhibitors currently used in clinical trials and those that exhibit promising results.
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Affiliation(s)
- Yannick Delpu
- Cancer Research Center of Toulouse Inserm UMR 1037, 31432 Toulouse cedex 4, France; E-Mails: (Y.D.); (P.C.)
- University de Toulouse III-Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse cedex 9, France
| | - Pierre Cordelier
- Cancer Research Center of Toulouse Inserm UMR 1037, 31432 Toulouse cedex 4, France; E-Mails: (Y.D.); (P.C.)
- University de Toulouse III-Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse cedex 9, France
| | - William C. Cho
- Department of Clinical Oncology, Queen Elizabeth Hospital, Hong Kong, China; E-Mail:
| | - Jérôme Torrisani
- Cancer Research Center of Toulouse Inserm UMR 1037, 31432 Toulouse cedex 4, France; E-Mails: (Y.D.); (P.C.)
- University de Toulouse III-Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse cedex 9, France
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Jin B, Robertson KD. DNA methyltransferases, DNA damage repair, and cancer. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2013; 754:3-29. [PMID: 22956494 DOI: 10.1007/978-1-4419-9967-2_1] [Citation(s) in RCA: 347] [Impact Index Per Article: 28.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The maintenance DNA methyltransferase (DNMT) 1 and the de novo methyltransferases DNMT3A and DNMT3B are all essential for mammalian development. DNA methylation, catalyzed by the DNMTs, plays an important role in maintaining genome stability. Aberrant expression of DNMTs and disruption of DNA methylation patterns are closely associated with many forms of cancer, although the exact mechanisms underlying this link remain elusive. DNA damage repair systems have evolved to act as a genome-wide surveillance mechanism to maintain chromosome integrity by recognizing and repairing both exogenous and endogenous DNA insults. Impairment of these systems gives rise to mutations and directly contributes to tumorigenesis. Evidence is mounting for a direct link between DNMTs, DNA methylation, and DNA damage repair systems, which provide new insight into the development of cancer. Like tumor suppressor genes, an array of DNA repair genes frequently sustain promoter hypermethylation in a variety of tumors. In addition, DNMT1, but not the DNMT3s, appear to function coordinately with DNA damage repair pathways to protect cells from sustaining mutagenic events, which is very likely through a DNA methylation-independent mechanism. This chapter is focused on reviewing the links between DNA methylation and the DNA damage response.
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Affiliation(s)
- Bilian Jin
- Department of Biochemistry and Molecular Biology, Georgia Health Sciences University Cancer Center, CN-2151, 1410 Laney Walker Blvd, Augusta, GA 30912, USA
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12
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Hile SE, Shabashev S, Eckert KA. Tumor-specific microsatellite instability: do distinct mechanisms underlie the MSI-L and EMAST phenotypes? Mutat Res 2012. [PMID: 23206442 DOI: 10.1016/j.mrfmmm.2012.11.003] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Microsatellite DNA sequences display allele length alterations or microsatellite instability (MSI) in tumor tissues, and MSI is used diagnostically for tumor detection and classification. We discuss the known types of tumor-specific MSI patterns and the relevant mechanisms underlying each pattern. Mutation rates of individual microsatellites vary greatly, and the intrinsic DNA features of motif size, sequence, and length contribute to this variation. MSI is used for detecting mismatch repair (MMR)-deficient tumors, which display an MSI-high phenotype due to genome-wide microsatellite destabilization. Because several pathways maintain microsatellite stability, tumors that have undergone other events associated with moderate genome instability may display diagnostic MSI only at specific di- or tetranucleotide markers. We summarize evidence for such alternative MSI forms (A-MSI) in sporadic cancers, also referred to as MSI-low and EMAST. While the existence of A-MSI is not disputed, there is disagreement about the origin and pathologic significance of this phenomenon. Although ambiguities due to PCR methods may be a source, evidence exists for other mechanisms to explain tumor-specific A-MSI. Some portion of A-MSI tumors may result from random mutational events arising during neoplastic cell evolution. However, this mechanism fails to explain the specificity of A-MSI for di- and tetranucleotide instability. We present evidence supporting the alternative argument that some A-MSI tumors arise by a distinct genetic pathway, and give examples of DNA metabolic pathways that, when altered, may be responsible for instability at specific microsatellite motifs. Finally, we suggest that A-MSI in tumors could be molecular signatures of environmental influences and DNA damage. Importantly, A-MSI occurs in several pre-neoplastic inflammatory states, including inflammatory bowel diseases, consistent with a role of oxidative stress in A-MSI. Understanding the biochemical basis of A-MSI tumor phenotypes will advance the development of new diagnostic tools and positively impact the clinical management of individual cancers.
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Affiliation(s)
- Suzanne E Hile
- Department of Pathology, Gittlen Cancer Research Foundation, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA
| | - Samion Shabashev
- Department of Pathology, Gittlen Cancer Research Foundation, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA
| | - Kristin A Eckert
- Department of Pathology, Gittlen Cancer Research Foundation, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA.
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13
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Campregher C, Schmid G, Ferk F, Knasmüller S, Khare V, Kortüm B, Dammann K, Lang M, Scharl T, Spittler A, Roig AI, Shay JW, Gerner C, Gasche C. MSH3-deficiency initiates EMAST without oncogenic transformation of human colon epithelial cells. PLoS One 2012; 7:e50541. [PMID: 23209772 PMCID: PMC3507781 DOI: 10.1371/journal.pone.0050541] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2012] [Accepted: 10/25/2012] [Indexed: 12/16/2022] Open
Abstract
BACKGROUND/AIM Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature in certain cases of sporadic colorectal cancer and has been linked to MSH3-deficiency. It is currently controversial whether EMAST is associated with oncogenic properties in humans, specifically as cancer development in Msh3-deficient mice is not enhanced. However, a mutator phenotype is different between species as the genetic positions of repetitive sequences are not conserved. Here we studied the molecular effects of human MSH3-deficiency. METHODS HCT116 and HCT116+chr3 (both MSH3-deficient) and primary human colon epithelial cells (HCEC, MSH3-wildtype) were stably transfected with an EGFP-based reporter plasmid for the detection of frameshift mutations within an [AAAG]17 repeat. MSH3 was silenced by shRNA and changes in protein expression were analyzed by shotgun proteomics. Colony forming assay was used to determine oncogenic transformation and double strand breaks (DSBs) were assessed by Comet assay. RESULTS Despite differential MLH1 expression, both HCT116 and HCT116+chr3 cells displayed comparable high mutation rates (about 4×10(-4)) at [AAAG]17 repeats. Silencing of MSH3 in HCECs leads to a remarkable increased frameshift mutations in [AAAG]17 repeats whereas [CA]13 repeats were less affected. Upon MSH3-silencing, significant changes in the expression of 202 proteins were detected. Pathway analysis revealed overexpression of proteins involved in double strand break repair (MRE11 and RAD50), apoptosis, L1 recycling, and repression of proteins involved in metabolism, tRNA aminoacylation, and gene expression. MSH3-silencing did not induce oncogenic transformation and DSBs increased 2-fold. CONCLUSIONS MSH3-deficiency in human colon epithelial cells results in EMAST, formation of DSBs and significant changes of the proteome but lacks oncogenic transformation. Thus, MSH3-deficiency alone is unlikely to drive human colon carcinogenesis.
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Affiliation(s)
- Christoph Campregher
- Christian Doppler Laboratory for Molecular Cancer Chemoprevention, Division of Gastroenterology and Hepatology, Department of Medicine 3, Medical University of Vienna, Vienna, Austria
| | - Gerald Schmid
- Christian Doppler Laboratory for Molecular Cancer Chemoprevention, Division of Gastroenterology and Hepatology, Department of Medicine 3, Medical University of Vienna, Vienna, Austria
| | - Franziska Ferk
- Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Vienna, Austria
| | - Siegfried Knasmüller
- Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Vienna, Austria
| | - Vineeta Khare
- Christian Doppler Laboratory for Molecular Cancer Chemoprevention, Division of Gastroenterology and Hepatology, Department of Medicine 3, Medical University of Vienna, Vienna, Austria
| | - Benedikt Kortüm
- Christian Doppler Laboratory for Molecular Cancer Chemoprevention, Division of Gastroenterology and Hepatology, Department of Medicine 3, Medical University of Vienna, Vienna, Austria
| | - Kyle Dammann
- Christian Doppler Laboratory for Molecular Cancer Chemoprevention, Division of Gastroenterology and Hepatology, Department of Medicine 3, Medical University of Vienna, Vienna, Austria
| | - Michaela Lang
- Christian Doppler Laboratory for Molecular Cancer Chemoprevention, Division of Gastroenterology and Hepatology, Department of Medicine 3, Medical University of Vienna, Vienna, Austria
| | - Theresa Scharl
- ACIB GmbH, c/o Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria
- Department of Statistics and Probability Theory, University of Technology, Vienna, Austria
| | - Andreas Spittler
- Department of Surgery, Research Laboratories & Core Facility Flow Cytometry, Medical University of Vienna, Vienna, Austria
| | - Andres I. Roig
- Department of Cell Biology, Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
| | - Jerry W. Shay
- Department of Cell Biology, Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
| | - Christopher Gerner
- Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria
| | - Christoph Gasche
- Christian Doppler Laboratory for Molecular Cancer Chemoprevention, Division of Gastroenterology and Hepatology, Department of Medicine 3, Medical University of Vienna, Vienna, Austria
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14
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Humans accumulate microsatellite instability with acquired loss of MLH1 protein in hematopoietic stem and progenitor cells as a function of age. Blood 2012; 120:3229-36. [PMID: 22740444 DOI: 10.1182/blood-2011-12-401950] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
Hematopoietic stem and progenitor cells (HPCs) are necessary for long-term survival. Genomic instability and persistent DNA damage may cause loss of adult stem cell function. The mismatch repair (MMR) pathway increases replication fidelity and defects have been implicated in malignant hematopoietic diseases. Little, however, is known about the role MMR pathway failure plays in the aging process of human HPCs. We hypothesized that loss of MMR occurs in HPCs as a process of human aging. We examined microsatellite instability and expression of the MMR genes MutL homologue 1 (MLH1) and MutS homologue 2 (MSH2) in HPCs and colony-forming cell-derived clones (CFCs) from human donors aged 0 to 86 years. CFCs from donors > 45 years had a greater frequency of microsatellite instability and CD34(+) progenitors lacking MLH1 expression and protein than individuals ≤ 45 years. Loss of MSH2 did not correlate with age. Thus, a potentially early event in the normal human aging process is microsatellite instability accumulation in normal human HPCs associated with the loss of MLH1 protein expression.
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Agenesis of the corpus callosum and gray matter heterotopia in three patients with constitutional mismatch repair deficiency syndrome. Eur J Hum Genet 2012; 21:55-61. [PMID: 22692065 DOI: 10.1038/ejhg.2012.117] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
Constitutional mismatch repair deficiency (CMMR-D) syndrome is a rare inherited childhood cancer predisposition caused by biallelic germline mutations in one of the four mismatch repair (MMR)-genes, MLH1, MSH2, MSH6 or PMS2. Owing to a wide tumor spectrum, the lack of specific clinical features and the overlap with other cancer predisposing syndromes, diagnosis of CMMR-D is often delayed in pediatric cancer patients. Here, we report of three new CMMR-D patients all of whom developed more than one malignancy. The common finding in these three patients is agenesis of the corpus callosum (ACC). Gray matter heterotopia is present in two patients. One of the 57 previously reported CMMR-D patients with brain tumors (therefore all likely had cerebral imaging) also had ACC. With the present report the prevalence of cerebral malformations is at least 4/60 (6.6%). This number is well above the population birth prevalence of 0.09-0.36 live births with these cerebral malformations, suggesting that ACC and heterotopia are features of CMMR-D. Therefore, the presence of cerebral malformations in pediatric cancer patients should alert to the possible diagnosis of CMMR-D. ACC and gray matter heterotopia are the first congenital malformations described to occur at higher frequency in CMMR-D patients than in the general population. Further systematic evaluations of CMMR-D patients are needed to identify possible other malformations associated with this syndrome.
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Kantelinen J, Kansikas M, Candelin S, Hampel H, Smith B, Holm L, Kariola R, Nyström M. Mismatch repair analysis of inherited MSH2 and/or MSH6 variation pairs found in cancer patients. Hum Mutat 2012; 33:1294-301. [PMID: 22581703 DOI: 10.1002/humu.22119] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2011] [Accepted: 04/19/2012] [Indexed: 12/24/2022]
Abstract
Mismatch repair (MMR) malfunction causes the accumulation of mismatches in the genome leading to genomic instability and cancer. The inactivation of an MMR gene (MSH2, MSH6, MLH1, or PMS2) with an inherited mutation causes Lynch syndrome (LS), a dominant susceptibility to cancer. MMR gene variants of uncertain significance (VUS) may be pathogenic mutations, which cause LS, may result in moderately increased cancer risks, or may be harmless polymorphisms. Our study suggests that an inherited MMR VUS individually assessed as proficient may, however, in a pair with another MMR VUS found in the same colorectal cancer (CRC) patient have a concomitant contribution to the MMR deficiency. Here, eight pairs of MMR gene variants found in cancer patients were functionally analyzed in an in vitro MMR assay. Although the other pairs do not suggest a compound deficiency, the MSH2 VUS pair c.380A>G/c.982G>C (p.Asn127Ser/p.Ala328Pro), which nearly halves the repair capability of the wild-type MSH2 protein, is presumed to increase the cancer risk considerably. Moreover, two MSH6 variants, c.1304T>C (p.Leu435Pro) and c.1754T>C (p.Leu585Pro), were shown to be MMR deficient. The role of one of the most frequently reported MMR gene VUS, MSH2 c.380A>G (p.Asn127Ser), is especially interesting because its concomitant defect with another variant could finally explain its recurrent occurrence in CRC patients.
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Affiliation(s)
- Jukka Kantelinen
- Division of Genetics, Department of Biosciences, University of Helsinki, Helsinki, Finland
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Laghi L, Bianchi P, Delconte G, Celesti G, Di Caro G, Pedroni M, Chiaravalli AM, Jung B, Capella C, de Leon MP, Malesci A. MSH3 protein expression and nodal status in MLH1-deficient colorectal cancers. Clin Cancer Res 2012; 18:3142-53. [PMID: 22496206 DOI: 10.1158/1078-0432.ccr-12-0175] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE Patients with colorectal cancers (CRC) and high microsatellite instability (MSI) have a better outcome than their chromosome-unstable counterpart. Given the heterogeneity of microsatellite-unstable CRCs, we wanted to see whether any MSI-associated molecular features are specifically associated with prognosis. EXPERIMENTAL DESIGN One hundred and nine MSI-high CRCs were typed for primary mismatch repair (MMR) defect and for secondary loss of MMR proteins. Frameshifts at seven target genes, mutations in the RAS pathway, and methylation at MLH1/CDKN2A promoters were also searched. The interplay of molecular findings with clinicopathologic features and patient survival was analyzed. RESULTS Of 84 MLH1-deficient CRCs, 31 (36.9%) had MSH3 and 11 (13.1%) had MSH6 loss (P < 0.001), biallelic frameshift mutations at mononucleotide repeats accounting for most (78%) MSH3 losses. As compared with MSH3-retaining cancers, MLH1-deficient tumors with MSH3 loss showed a higher number of mutated target genes (3.94 ± 1.56 vs. 2.79 ± 1.75; P = 0.001), absence of nodal involvement at pathology [N0; OR, 0.11; 95% confidence interval (CI), 0.04-0.43, P < 0.001], and better disease-free survival (P = 0.06). No prognostic value was observed for KRAS status and for MLH1/CDKN2A promoter methylation. The association between MSH3 loss and N0 was confirmed in an independent cohort of 71 MLH1-deficient CRCs (OR, 0.23; 95% CI, 0.06-0.83, P = 0.02). CONCLUSIONS MLH1-deficient CRCs not expressing MSH3 have a more severe MSI, a lower rate of nodal involvement, and a better postsurgical outcome.
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Affiliation(s)
- Luigi Laghi
- Laboratory of Molecular Gastroenterology, Department of Gastroenterology, IRCCS Istituto Clinico Humanitas - Rozzano, Milano, Italy.
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A putative Lynch syndrome family carrying MSH2 and MSH6 variants of uncertain significance-functional analysis reveals the pathogenic one. Fam Cancer 2012; 10:515-20. [PMID: 21431882 DOI: 10.1007/s10689-011-9436-z] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
Inherited pathogenic mutations in the mismatch repair (MMR) genes, MSH2, MLH1, MSH6, and PMS2 predispose to Lynch syndrome (LS). However, the finding of a variant or variants of uncertain significance (VUS) in affected family members complicates the risk assessment. Here, we describe a putative LS family carrying VUS in both MSH2 (c.2768T>A, p.Val923Glu) and MSH6 (c.3563G>A, p.Ser1188Asn). Two colorectal cancer (CRC) patients were studied for mutations and identified as carriers of both variants. In spite of a relatively high mean age of cancer onset (59.5 years) in the family, many CRC patients and the tumor pathological data suggested that the missense variation in MSH2, the more common susceptibility gene in LS, would be the predisposing alteration. However, MSH2 VUS was surprisingly found to be MMR proficient in an in vitro MMR assay and a tolerant alteration in silico. By supplying evidence that instead of MSH2 p.Val923Glu the MSH6 p.Ser1188Asn variant is completely MMR-deficient, the present study confirms the previous findings, and suggests that MSH6 (c.3563G>A, p.Ser1188Asn) is the pathogenic mutation in the family. Moreover, our results strongly support the strategy to functionally assess all identified VUS before predictive gene testing and genetic counseling are offered to a family.
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Loughery JEP, Dunne PD, O'Neill KM, Meehan RR, McDaid JR, Walsh CP. DNMT1 deficiency triggers mismatch repair defects in human cells through depletion of repair protein levels in a process involving the DNA damage response. Hum Mol Genet 2011; 20:3241-55. [PMID: 21636528 DOI: 10.1093/hmg/ddr236] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/01/2025] Open
Abstract
DNA methyltransferase 1 (DNMT1) maintains methylation at CpG dinucleotides, important for transcriptional silencing at many loci. It is also implicated in stabilizing repeat sequences: DNMT1 deficiency causes microsatellite instability in mouse embryonic stem cells, but it is unclear how this occurs, how repeats lacking CpG become unstable and whether the effect is confined to stem cells. To address these questions, we transfected hTERT-immortalized normal human fibroblasts (hTERT-1604) with a short hairpin RNA construct targeting DNMT1 and isolated stable integrants with different levels of protein. DNMT1 expression levels agreed well with methylation levels at imprinted genes. Knockdown cells showed two key characteristics of mismatch repair (MMR) deficiency, namely resistance to the drug 6-thioguanine and up to 10-fold elevated mutation rates at a CA(17) microsatellite reporter, but had limited viability. The likely cause of MMR defects is a matching drop in steady-state protein levels for key repair components in DNMT1 knockdown cells, affecting both the MutLα and MutSα complexes. This indirect effect on MMR proteins was also seen using a different targeting method in HT29 colon cancer cells and did not involve transcriptional silencing of the respective genes. Decreased levels of MMR components follow activation of the DNA damage response and blocking this response, and in particular poly(ADP-ribose) polymerase (PARP) overactivation, rescues cell viability in DNMT1-depleted cells. These results offer an explanation for how and why unmethylated microsatellite repeats can be destabilized in cells with decreased DNMT1 levels and uncover a novel and important role for PARP in this process.
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Affiliation(s)
- Jayne E P Loughery
- Transcriptional Regulation and Epigenetics Group, Centre for Molecular Biosciences, University of Ulster, Coleraine BT52 1SA, UK
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