Complete loss or deletion of the long arm of chromosome 5 is frequent in myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). The putative gene(s) deleted and responsible for the pathogenesis of these poor prognosis hematologic disorders remain controversial. This study is a comprehensive analysis of previously implicated and novel genes for epigenetic inactivation in AML and MDS. In 146 AML cases, methylation of CTNNA1 was frequent, and more common in AML patients with 5q deletion (31%) than those without 5q deletion (14%), whereas no methylation of other 5q genes was observed. In 31 MDS cases, CTNNA1 methylation was only found in high-risk MDS (>or=RAEB2), but not in low-risk MDS (
Tur MK, Neef I, Jost E, et al.Targeted restoration of down-regulated DAPK2 tumor suppressor activity induces apoptosis in Hodgkin lymphoma cells.
J Immunother. 2009; 32(5):431-41 [PubMed
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Death-associated protein kinase 2 (DAPK2) is a calcium/calmodulin-regulated proapoptotic serine/threonine kinase that acts as a tumor suppressor. Here we show that DAPK2 is down-regulated in Hodgkin lymphoma-derived tumor cell lines and that promoter-region hypermethylation is one mechanism for DAPK2 inactivation. To determine whether selective reconstitution of DAPK2 catalytic activity in these cells could induce apoptosis, we created a fusion protein comprising a human CD30 ligand conjugated to a human DAPK2 calmodulin-deletion mutant. Thus, recombinant immunokinase DAPK2'-CD30L has a constitutive kinase activity with enhanced proapoptotic function. We show that this immunokinase fusion protein inhibits cell proliferation and induces apoptotic cell death specifically in CD30/DAPK2-negative tumor cell lines. This proof-of-concept study provides the first demonstration of therapeutic strategies based on the restoration of a defective, tumor-suppressing kinase activity by a novel class of recombinant immunotherapeutics.
INTRODUCTION: Endothelin (EDN) signalling plays a crucial role in cell differentiation, proliferation and migration processes. There is compelling evidence that altered EDN signalling is involved in carcinogenesis by modulating cell survival and promoting invasiveness. To date, most reports have focused on the oncogenic potential of EDN1 and EDN2, both of which are overexpressed in various tumour entities. Here, we aimed at a first comprehensive analysis on EDN3 expression and its implication in human breast cancer.
METHODS: EDN3 mRNA expression was assessed by Northern blotting in normal human tissues (n = 9) as well as in matched pairs of normal and tumourous tissues from breast specimens (n = 50). EDN3 mRNA expression in breast cancer was further validated by real-time polymerase chain reaction (PCR) (n = 77). A tissue microarray was used to study EDN3 protein expression in breast carcinoma (n = 150) and normal breast epithelium (n = 44). EDN3 promoter methylation was analysed by methylation-specific PCR in breast cell lines (n = 6) before and after demethylating treatment, normal breast tissues (n = 17) and primary breast carcinomas (n = 128). EDN3 expression and methylation data were statistically correlated with clinical patient characteristics and patient outcome.
RESULTS: Loss of EDN3 mRNA expression in breast cancer, as initially detected by array-based expression profiling, could be confirmed by Northern blot analysis (> 2-fold loss in 96%) and real-time PCR (> 2-fold loss in 78%). Attenuated EDN3 expression in breast carcinoma was also evident at the protein level (45%) in association with adverse patient outcome in univariate (P = 0.022) and multivariate (hazard ratio 2.0; P = 0.025) analyses. Hypermethylation of the EDN3 promoter could be identified as the predominant mechanism leading to gene silencing. Reversion of the epigenetic lock by 5-aza-2'-deoxycytidine and trichostatin A resulted in EDN3 mRNA re-expression in vitro. Furthermore, EDN3 promoter hypermethylation was detected in 70% of primary breast carcinomas with significant association to loss of EDN3 mRNA expression (P = 0.005), whilst normal matched breast tissues revealed no EDN3 promoter methylation.
CONCLUSIONS: EDN3 is a frequent target of epigenetic inactivation in human breast cancer, potentially contributing to imbalanced EDN signalling commonly found in this disease. The clinical implication supports the view that EDN3, in contrast to EDN1 and EDN2, may act as natural tumour suppressor in the human mammary gland.
BACKGROUND: Inhibitor of differentiation 4 (Id4), a member of the Id gene family is also a dominant negative regulator of basic helix loop helix (bHLH) transcription factors. Some of the functions of Id4 appear to be unique as compared to its other family members Id1, Id2 and Id3. Loss of Id4 gene expression in many cancers in association with promoter hypermethylation has led to the proposal that Id4 may act as a tumor suppressor. In this study we provide functional evidence that Id4 indeed acts as a tumor suppressor and is part of a cancer associated epigenetic re-programming.
METHODS: Data mining was used to demonstrate Id4 expression in prostate cancer. Methylation specific polymerase chain reaction (MSP) analysis was performed to understand molecular mechanisms associated with Id4 expression in prostate cancer cell lines. The effect of ectopic Id4 expression in DU145 cells was determined by cell cycle analysis (3H thymidine incorporation and FACS), expression of androgen receptor, p53 and cyclin dependent kinase inhibitors p27 and p21 by a combination of RT-PCR, real time-PCR, western blot and immuno-cytochemical analysis.
RESULTS: Id4 expression was down-regulated in prostate cancer. Id4 expression was also down-regulated in prostate cancer line DU145 due to promoter hyper-methylation. Ectopic Id4 expression in DU145 prostate cancer cell line led to increased apoptosis and decreased cell proliferation due in part by an S-phase arrest. In addition to S-phase arrest, ectopic Id4 expression in PC3 cells also resulted in prolonged G2/M phase. At the molecular level these changes were associated with increased androgen receptor (AR), p21, p27 and p53 expression in DU145 cells.
CONCLUSION: The results suggest that Id4 acts directly as a tumor suppressor by influencing a hierarchy of cellular processes at multiple levels that leads to a decreased cell proliferation and change in morphology that is possibly mediated through induction of previously silenced tumor suppressors.
Seeliger B, Wilop S, Osieka R, et al.CpG island methylation patterns in chronic lymphocytic leukemia.
Leuk Lymphoma. 2009; 50(3):419-26 [PubMed
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Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries. In CLL, a large number of genes affecting cancer-related pathways may be dysregulated by epigenetic silencing. We analysed by methylation-specific polymerase chain reaction the CpG island methylation status of 15 well-characterised cancer-related genes in 32 patients with CLL. Aberrant methylation in the sample of patients with CLL was shown for secreted frizzled-related protein 1 (68.8%), secreted frizzled-related protein 2 (65.6%), death-associated protein kinase 1 (50.0%), E-cadherin (21.9%), secreted frizzled-related protein 4 (15.6%), p15 (9.4%), p16 (6.3%), retinoic acid receptor beta2 (3.1%), secreted frizzled-related protein 5 (3.1%) and tissue inhibitor of matrix metalloproteinases 3 (3.1%). For human Mut-L homolog 1, O(6)-methylguanine DNA methyltransferase, p73, suppressor of cytokine signalling 1 and tissue inhibitor of matrix metalloproteinases 2 no hypermethylation was detected. Hypermethylation of at least one gene was observed in 87.5% of the samples. Our results show that aberrant CpG island methylation affecting cancer-related pathways such as Wnt signalling, regulation of apoptosis, cell cycle control and tissue invasion is a common phenomenon in CLL. Epigenetic disturbances may be involved in the pathogenesis of CLL and thus may provide a molecular rationale for therapeutic approaches.
Jost E, Gezer D, Wilop S, et al.Epigenetic dysregulation of secreted Frizzled-related proteins in multiple myeloma.
Cancer Lett. 2009; 281(1):24-31 [PubMed
] Related Publications
We analysed the clinical impact of epigenetic dysregulation of the Wnt pathway in malignant plasma cell disorders. In multiple myeloma (MM) cell lines, aberrant promoter hypermethylation of the secreted Frizzled-related protein (SFRP) genes was a common event, and hypermethylation of SFRP1,-2 and -5 was associated with transcriptional silencing. Among 76 primary patient samples, the frequency of aberrant methylation was 35.5% for SFRP1, 52.6% for SFRP2, 1.3% for SFRP4 and 6.9% for SFRP5. Hypermethylation of SFRP1 and -2 genes was detected in monoclonal gammopathy of undetermined significance and all MM stages including plasma cell leukaemia (PCL), while SFRP5 methylation was restricted to advanced MM stages and PCL. Our data indicate that epigenetic silencing of Wnt antagonists is an early event in MM pathogenesis and that SFRP5 hypermethylation may play a role in disease progression.
Jost E, do O N, Wilop S, et al.Aberrant DNA methylation of the transcription factor C/EBPalpha in acute myelogenous leukemia.
Leuk Res. 2009; 33(3):443-9 [PubMed
] Related Publications
We investigated the methylation status of the CCAAT/enhancer binding protein alpha (C/EBPalpha) promoter region near the transcription start site in acute myelogenous leukemia (AML). In hematopoietic tumor cell lines, CpG island hypermethylation of the proximal C/EBPalpha promoter region was associated with transcriptional silencing, and treatment with the demethylating agent 5-aza-2'-deoxycytidine resulted in C/EBPalpha reexpression and promoter demethylation. Aberrant methylation of the C/EBPalpha promoter region occurred in 10/80 diagnostic AML samples, and there was an inverse correlation between aberrant methylation of C/EBPalpha and the negative cell cycle regulator p15. Our results provide further evidence for epigenetic dysregulation of C/EBPalpha in AML.
Jost E, Schmid J, Wilop S, et al.Epigenetic inactivation of secreted Frizzled-related proteins in acute myeloid leukaemia.
Br J Haematol. 2008; 142(5):745-53 [PubMed
] Related Publications
The Wnt signalling pathway has a key function in stem cell maintenance and differentiation of haematopoietic progenitors. Secreted Frizzled-related protein genes (SFRPs), functioning as Wnt signalling antagonists, have been found to be downregulated by promoter hypermethylation in many tumours. To analyse epigenetic dysregulation of SFRPs in acute myeloid leukaemia (AML), we examined the promoter methylation status of SFRP1, -2, -4 and -5 in AML cell lines by methylation-specific polymerase chain reaction (MSP). Aberrant CpG island methylation was found for all four SFRP genes. By real-time reverse transcription-PCR, corresponding transcriptional silencing for SFRP1 and -2 was demonstrated and treatment of cell lines with 5-aza-2'-deoxycytidine resulted in re-expression. The methylation status of the SFRP genes was analysed in 100 specimens obtained from AML patients at diagnosis. The frequencies of aberrant methylation among the patient samples were 29% for SFRP1, 19% for SFRP2, 0% for SFRP4 and 9% for SFRP5. For SFRP2, a correlation between promoter hypermethylation and transcriptional downregulation was found in primary AML samples. Among AML cases with a favourable karyotype, hypermethylation of SFRP genes was restricted to patients with core binding factor (CBF) leukaemia, and aberrant methylation of the SFRP2 promoter was an adverse risk factor for survival in CBF leukaemia.
BACKGROUND: Inhibitor of DNA binding/Inhibitor of differentiation 4 (ID4) is a critical factor for cell proliferation and differentiation in normal vertebrate development. ID4 has regulative functions for differentiation and growth of the developing brain. The role of ID1, ID2 and ID3 are expected to be oncogenic due to their overexpression in pancreatic cancer and colorectal adenocarcinomas, respectively. Aside from these findings, loss of ID3 expression was demonstrated in ovarian cancer. The aim of the present study was to reveal the factual role of ID4 in carcinogenesis in more detail, since its role for the pathogenesis of human breast cancer has been discussed controversially, assigning both oncogenic and tumour suppressive functions.
METHODS: ID4 promoter methylation, ID4 mRNA expression and ID4 protein expression were analysed in primary human breast cancer specimens using methylation-specific PCR (MSP) (n=170), semiquantitative realtime RT-PCR (n=46) and immunhistochemistry (n=3), respectively. In order to demonstrate a functional association of ID4 promoter methylation with its gene silencing, we performed DNA demethylation analysis with four human breast cell lines using MSP and semiquantitative realtime RT-PCR. In addition, we performed correlations of ID4 promoter methylation with ID4 mRNA and ID4 protein expression in matched samples of breast tumour and corresponding normal tissue. We carried out statistical analyses in order to find correlations between ID4 promoter methylation and clinicopathological parameters.
RESULTS: Frequent ID4 promoter methylation was observed in primary breast cancer samples (69%, 117/170). We found a tight correlation (P<0.0001) between ID4 promoter methylation and loss of ID4 expression in primary breast cancer 3 specimens. Demethylating treatment with breast cancer cell lines was associated with clear ID4 mRNA re-expression. Tumours with ID4 promoter methylation showed distinct loss of ID4 expression on both transcription and protein level. Interestingly, ID4 promoter methylation was a factor for unfavourable recurrence-free survival (P=0.036) and increased risk for lymph node metastasis (P=0.030).
CONCLUSION: ID4 is indeed a novel tumour suppressor gene in normal human breast tissue and is epigenetically silenced during cancer development, indicating increased risk for tumour relapse. Thus, ID4 methylation status could serve as a prognostic biomarker in human breast cancer.
Jost E, Galm OEHA scientific workshop report: the role of epigenetics in hematological malignancies.
Epigenetics. 2007 Apr-Jun; 2(2):71-9 [PubMed
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The clinical application of targeting the epigenome in cancer cells through demethylation and histone acetylation is an exiting novel pharmacologic concept in the treatment of malignant diseases. The manipulation of gene expression through epigenetic modifications represents a new strategy in targeted anti-cancer therapy. A workshop was held in Cannes, France in February 2007 to discuss the latest findings in basic and clinical research regarding the role of epigenetics in haematological malignancies. Fundamental aspects of DNA methylation, the histone code, chromatin remodelling and transcriptional regulatory mechanisms were discussed. Recent technological advances are now allowing scientists to gain further insight in the altered epigenome during tumorigenesis and will provide numerous opportunities for translational research. The aim of this article is to summarize the major topics discussed at the workshop by leading experts in the field and highlight conclusions for future basic, translational and clinical research.
Jost E, do O N, Dahl E, et al.Epigenetic alterations complement mutation of JAK2 tyrosine kinase in patients with BCR/ABL-negative myeloproliferative disorders.
Leukemia. 2007; 21(3):505-10 [PubMed
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An acquired autoactivating mutation with a V617F amino-acid substitution in the JAK2 tyrosine kinase is frequently found in BCR/ABL-negative myeloproliferative disorders (MPD). Hypermethylation of CpG islands within gene promoter regions is associated with transcriptional inactivation and represents an important mechanism of gene silencing in the pathogenesis of hematopoietic malignancies. In this study, we determined the DNA methylation status of 13 cancer-related genes in the context of JAK2 mutations in 39 patients with MPD. Genes analyzed for hypermethylation were SOCS-1, SHP-1, E-cadherin, MGMT, TIMP-2, TIMP-3, p15, p16, p73, DAPK1, RASSF1A, RARbeta2 and hMLH1. We found at least one hypermethylated gene in 15/39 MPD patient specimens, and in 6/39 samples aberrant methylation of the negative cytokine regulator SOCS-1 was present. The JAK2V617F mutation was found in 21/39 patients as determined by allele-specific polymerase chain reaction. Hypermethylation of SOCS-1 was observed in 3/21 patients with an autoactivating JAK2 mutation and in 3/18 patients with wild-type JAK2. Our results suggest that epigenetic inactivation of SOCS-1 may be a complementary mechanism to the JAK2V617F mutation in the pathogenesis of MPD that leads to dysregulation of JAK-STAT signal transduction and thus contributes to growth factor hypersensitivity.
Gore SD, Baylin S, Sugar E, et al.Combined DNA methyltransferase and histone deacetylase inhibition in the treatment of myeloid neoplasms.
Cancer Res. 2006; 66(12):6361-9 [PubMed
] Related Publications
Optimal reexpression of most genes silenced through promoter methylation requires the sequential application of DNA methyltransferase inhibitors followed by histone deacetylase inhibitors in tumor cell cultures. Patients with myelodysplastic syndrome or acute myeloid leukemia (AML) were treated with the methyltransferase inhibitor 5-azacitidine (aza-CR) followed by the histone deacetylase inhibitor sodium phenylbutyrate. Major responses associated with cytogenetic complete response developed in patients receiving prolonged dosing schedules of aza-CR. Bisulfite sequencing of the p15 promoter in marrow DNA during the first cycle of treatment showed heterogeneous allelic demethylation in three responding patients, suggesting ongoing demethylation within the tumor clone, but no demethylation in two nonresponders. Six of six responding patients with pretreatment methylation of p15 or CDH-1 promoters reversed methylation during the first cycle of therapy (methylation-specific PCR), whereas none of six nonresponders showed any demethylation. Gene demethylation correlated with the area under the aza-CR plasma concentration-time curve. Administration of both drugs was associated with induction of acetylation of histones H3 and H4. This study provides the first demonstration that molecular mechanisms responsible for responses to DNA methyltransferase/histone deacetylase inhibitor combinations may include reversal of aberrant epigenetic gene silencing. The promising percentage of major hematologic responses justifies the testing of such combinations in prospective randomized trials.
Veeck J, Niederacher D, An H, et al.Aberrant methylation of the Wnt antagonist SFRP1 in breast cancer is associated with unfavourable prognosis.
Oncogene. 2006; 25(24):3479-88 [PubMed
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The canonical Wnt signalling pathway plays a key role during embryogenesis and defects in this pathway have been implicated in the pathogenesis of various types of tumours, including breast cancer. The gene for secreted frizzled-related protein 1 (SFRP1) encodes a soluble Wnt antagonist and is located in a chromosomal region (8p22-p12) that is often deleted in breast cancer. In colon, lung, bladder and ovarian cancer SFRP1 expression is frequently inactivated by promoter methylation. We have previously shown that loss of SFRP1 protein expression is a common event in breast tumours that is associated with poor overall survival in patients with early breast cancer. To investigate the cause of SFRP1 loss in breast cancer, we performed mutation, methylation and expression analysis in human primary breast tumours and breast cell lines. No SFRP1 gene mutations were detected. However, promoter methylation of SFRP1 was frequently observed in both primary breast cancer (61%, n=130) and cell lines analysed by methylation-specific polymerase chain reaction (MSP). We found a tight correlation (P<0.001) between methylation and loss of SFRP1 expression in primary breast cancer tissue. SFRP1 expression was restored after treatment of tumour cell lines with the demethylating agent 5-aza-2'-deoxycytidine. Most interestingly, SFRP1 promoter methylation was an independent factor for adverse patient survival in Kaplan-Meier analysis. Our results indicate that promoter hypermethylation is the predominant mechanism of SFRP1 gene silencing in human breast cancer and that SFRP1 gene inactivation in breast cancer is associated with unfavourable prognosis.
Galm O, Herman JG, Baylin SBThe fundamental role of epigenetics in hematopoietic malignancies.
Blood Rev. 2006; 20(1):1-13 [PubMed
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The term epigenetics defines a heritable alteration in gene expression without an accompanying change in primary DNA sequence. Two major mechanisms that foster epigenetic changes are DNA methylation at cytosine bases within a CpG dinucleotide and post-translational histone modifications. Disruption of the balanced epigenetic network may have significant impact on chromatin structure and transcriptional activity. DNA methylation patterns are profoundly deranged in human cancer and comprise genome-wide losses as well as regional gains in DNA methylation. Hypermethylation of CpG islands within gene promoter regions in collaboration with deacetylation and other modifications of key histone amino acids is associated with transcriptional inactivation and represents, in addition to genetic aberrations, an important mechanism of gene silencing in the pathogenesis of human cancer. These epigenetic events act as alternatives to mutations and deletions to disrupt tumor suppressor gene function. A large number of genes involving fundamental cellular pathways may be affected by aberrant CpG island methylation in association with transcriptional silencing in virtually all tumor types. Altered DNA methylation patterns may serve as biomarkers for cancer detection, assessment of prognosis, and prediction of response to therapy. Furthermore, clinical trials using epigenetically targeted therapies have yielded promising results in hematopoietic malignancies. The ongoing exploration of basic events involved in altered gene transcription patterns and continued clinical investigative studies are helping to develop novel strategies for the diagnosis, prevention, and treatment of human cancer.
Galm O, Wilop S, Lüders C, et al.Clinical implications of aberrant DNA methylation patterns in acute myelogenous leukemia.
Ann Hematol. 2005; 84 Suppl 1:39-46 [PubMed
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Hypermethylation of CpG islands near gene promoter regions is associated with transcriptional inactivation and represents an important mechanism of gene silencing in carcinogenesis. Such epigenetic phenomena can act alongside DNA mutations and deletions to disrupt tumor-suppressor gene function. The methylation status of the promoter-associated CpG islands from 11 well-characterized cancer-related genes was analyzed by methylation-specific polymerase chain reaction in 60 adult patients with acute myelogenous leukemia (AML) at diagnosis. The frequency of aberrant methylation among the patient samples was 45.0% (27/60) for suppressor of cytokine signaling-1, 31.7% (19/60) for p15, 20.0% (12/60) for retinoic acid receptor beta2, 13.3% (8/60) for p73 and E-cadherin, 5.0% (3/60) for O(6)-methylguanine DNA methyltransferase, 3.3% (2/60) for death-associated protein kinase 1 and hMLH1, 1.7% (1/60) for p16, and 0% (0/60) for the tissue inhibitor of matrix metalloproteinases-3 and Ras association domain family 1A. Aberrant DNA methylation was found in AML of all French-American-British subtypes and throughout all cytogenetic risk groups. There appeared to be a trend towards a higher methylation frequency in AML patients with an unfavorable karyotype, but this difference was not statistically significant. Our data indicate that hypermethylation of multiple genes involving fundamental cellular pathways is a common event in AML, which varies greatly in frequency among the genes examined. The accumulation of epigenetic events affecting genes which are involved in regulating cell cycle inhibition, cell adhesion, growth factor signaling, and apoptosis may contribute to the malignant AML phenotype. The growing knowledge of the role of epigenetics in the aberrant silencing of cancer-related genes provides a rationale and molecular basis for targeted therapeutic approaches with demethylating agents in AML.
Methylation-specific polymerase chain reaction (MSP) is a method that can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails the initial modification of DNA by sodium bisulfite, converting all unmethylated cytosines to uracils but leaving the methylated cytosines unchanged, followed by subsequent amplification with primers specific for methylated vs unmethylated DNA. The great sensitivity of this technique allows qualitative methylation analysis from DNA obtained not only from fresh frozen tissues, peripheral blood, bone marrow, or body fluids but also from paraffin-embedded samples. It is a rapid and cost-effective method that does not require radioactive reagents and can be used for the analysis of a large number of clinical samples.
Galm O, Suzuki H, Akiyama Y, et al.Inactivation of the tissue inhibitor of metalloproteinases-2 gene by promoter hypermethylation in lymphoid malignancies.
Oncogene. 2005; 24(30):4799-805 [PubMed
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The tissue inhibitor of metalloproteinases-2 (TIMP-2) is known to antagonize matrix metalloproteinase activity and to suppress tumor growth, angiogenesis, invasion and metastasis. We analysed the methylation status of the CpG island in the TIMP-2 promoter region by methylation-specific polymerase chain reaction (MSP) in hematopoietic cell lines. TIMP-2 promoter hypermethylation in the lymphoma cell line Raji and the leukemia cell line KG1a was associated with transcriptional repression. Treatment with the demethylating agent 5-aza-2'-deoxycytidine resulted in TIMP-2 upregulation in both cell lines. TIMP-2 was expressed in the cell lines HL60, U266 and XG1, which carry an unmethylated promoter region. MSP analysis of primary patient samples revealed aberrant methylation of TIMP-2 in 33/90 (36.7%) cases of non-Hodgkin's lymphoma (NHL), but not in normal peripheral blood lymphocytes as well as in nonmalignant bone marrow and lymph nodes. The frequency of TIMP-2 methylation was slightly higher in aggressive NHL subtypes compared to those with an indolent subtype (38.6 versus 33.3%). In contrast, TIMP-2 was not hypermethylated in any of the 40 cases of acute myelogenous leukemia examined. We conclude that promoter hypermethylation of TIMP-2 is a novel epigenetic event in the pathogenesis of lymphoid malignancies and may contribute to a more aggressive NHL phenotype.
Lynch HT, Brand RE, Locker GYInflammatory bowel disease in Ashkenazi Jews: implications for familial colorectal cancer.
Fam Cancer. 2004; 3(3-4):229-32 [PubMed
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Inflammatory bowel disease (IBD) has a multifactorial etiology and includes ulcerative colitis (UC) and Crohn's disease (CD). Powerful epidemiologic and genetic studies have provided ample evidence that a subset of both CD and UC are attributable to a likely primary genetic etiology. This is evidenced by the recent identification of the IBD1 gene ( NOD2 ) mutations which show an association with susceptibility to CD. The IBD complex shows a significant increased frequency in Jews when compared to non-Jews. While there is an increased incidence of colorectal cancer (CRC) in patients with IBD, it nevertheless is important to realize that IBD likely accounts for no more than 1-3% of all cases of CRC in Ashkenazi Jews. Importantly, however, awareness of the increased CRC risk in IBD may aid immeasurably in preventive interventions. The molecular pathway leading to CRC in IBD appears to differ from the well-known adenoma-to-CRC sequence, given the fact that these cancers appear to arise from either flat, dysplastic tissue or dysplasia-associated lesions or masses (DALMs). An important model, but by no means an absolute one, for colon carcinogenesis in IBD follows progression from an absence of dysplasia, to indefinite dysplasia, to low-grade dysplasia, on to high-grade dysplasia, and ultimately to invasive CRC. This carcinogenic process relates to the disease duration with respect to the extent of colonic involvement and may also involve primary sclerosing cholangitis. Given this knowledge of an increased risk for CRC in UC and CD, surveillance colonoscopy should initially be performed 8-10 years after onset of symptoms as opposed to diagnosis, and it should be performed 1-2 years after 8 years of disease in patients with pancolitis or after 15 years in those with left-sided colitis. A search for dysplasia of colonic mucosa with biopsies performed in all four quadrants every 10 cm throughout the colon is exceedingly important. Additional biopsies should be taken of any flat lesions, masses, or strictures. Prophylactic colectomy may then be indicated when severe dysplasia is confirmed by knowledgeable pathologists.
Galm O, Esteller MBeyond genetics--the emerging role of epigenetic changes in hematopoietic malignancies.
Int J Hematol. 2004; 80(2):120-7 [PubMed
] Related Publications
The term epigenetic refers to a heritable change in gene expression that is mediated by mechanisms other than alterations in the primary nucleotide sequence. DNA methylation at cytosine bases that are located 5' to guanosine within a CpG dinucleotide is the main epigenetic modification in humans. Patterns of DNA methylation are profoundly deranged in human cancer and comprise genome-wide losses as well as regional gains in DNA methylation. Hypermethylation of CpG islands within gene promoter regions is associated with transcriptional inactivation and represents, in addition to genetic aberrations, an important mechanism of gene silencing in the pathogenesis of hematopoietic malignancies. This epigenetic phenomenon acts as an alternative to mutations and deletions to disrupt tumor suppressor gene function. A large number of genes involving fundamental cellular pathways may be affected in virtually all types of human cancer by aberrant CpG island methylation in association with transcriptional silencing. Altered methylation patterns can be used as biomarkers for cancer detection, assessment of prognosis, and prediction of response to antitumor treatment. Furthermore, clinical trials using epigenetically targeted therapies have yielded promising results for acute and chronic leukemias as well as for myelodysplastic syndromes. The exploration of our growing knowledge about epigenetic aberrations may help develop novel strategies for the diagnosis and treatment of hematopoietic malignancies in the future.
Galm O, Wilop S, Reichelt J, et al.DNA methylation changes in multiple myeloma.
Leukemia. 2004; 18(10):1687-92 [PubMed
] Related Publications
Using a candidate gene approach, we analyzed the methylation status of the promoter-associated CpG islands of 11 well-characterized tumor suppressor genes by methylation-specific polymerase chain reaction in five multiple myeloma (MM) cell lines and 56 patients with malignant plasma cell disorders. The frequency of aberrant methylation among the patient samples was 46.4% for SOCS-1, 35.7% for p16, 21.4% for E-cadherin, 12.5% for DAP kinase and p73, 1.8% for p15, MGMT as well as RARbeta, and 0% for TIMP-3, RASSF1A and hMLH1. We found at least one hypermethylated gene in 80.4% of the primary patient samples, while 33.9% harbored two or more hypermethylated genes. For the first time, we show that p73 may be hypermethylated in MM and thus be involved in the pathogenesis of plasma cell disorders. Hypermethylation of p16 at diagnosis was associated with a poorer prognosis. In patients with plasma cell leukemia, we found frequent simultaneous hypermethylation of p16, E-cadherin and DAP kinase. We conclude that aberrant methylation of tumor suppressor genes is a common event in malignant plasma cell disorders and that there is a correlation between methylation patterns and clinical characteristics in MM patients.
Galm O, Yoshikawa H, Esteller M, et al.SOCS-1, a negative regulator of cytokine signaling, is frequently silenced by methylation in multiple myeloma.
Blood. 2003; 101(7):2784-8 [PubMed
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The suppressor of cytokine signaling (SOCS) family of proteins has been implicated in the negative regulation of several cytokine pathways, particularly the receptor-associated tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathways of transcriptional activation. SOCS-1 (also known as JAB and SSI-1) inhibits signaling by many cytokines. Because of the previously observed hypermethylation-associated inactivation of SOCS-1 in hepatocellular carcinoma and the critical role of interleukin-6 (IL-6) as a survival factor in multiple myeloma (MM), we examined CpG island methylation of the SOCS-1 gene in MM cell lines and primary MM samples. Aberrant SOCS-1 methylation was found in the IL-6-dependent MM cell lines U266 and XG1, which correlated with transcriptional silencing. Treatment of these cell lines with the demethylating agent 5-aza-2'-deoxycytidine (DAC) up-regulated SOCS-1 expression. Methylation-associated inactivation of SOCS-1 in hematopoietic cell lines correlated with greater sensitivity to the chemical JAK inhibitor AG490. Using methylation-specific polymerase chain reaction (MSP), we found that SOCS-1 is hypermethylated in 62.9% (23/35) of MM patient samples. In contrast, methylation analysis of malignant lymphomas of various histologies revealed SOCS-1 hypermethylation in only 3.2% (2/62), and there was no methylation of SOCS-1 in normal peripheral blood leukocytes or bone marrow cells. We conclude that SOCS-1 is frequently inactivated by hypermethylation in MM patients. Silencing of the SOCS-1 gene may impair negative regulation of the Jak/STAT pathway and therefore result in greater responsiveness to cytokines, thus supporting survival and expansion of MM cells.
Esteller M, Guo M, Moreno V, et al.Hypermethylation-associated Inactivation of the Cellular Retinol-Binding-Protein 1 Gene in Human Cancer.
Cancer Res. 2002; 62(20):5902-5 [PubMed
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The effects of retinol (vitamin A) depend on its transport and binding to nuclear receptors. The cellular retinol-binding protein 1 (CRBP1) and the retinoic acid receptor beta2 (RARbeta2) are key components of this process. Loss of CRBP1 expression occurs in breast tumors, but the mechanism is not known. We examined whether CpG island hypermethylation of CRBP1 was responsible for its inactivation in cancer cell lines (n = 36) and primary tumors (n = 553) and its relation to RARbeta2 methylation. Hypermethylation of CRBP1 was common in tumors and cancer cell lines, with the highest frequency in lymphoma and gastrointestinal carcinomas. Hypermethylation correlated with loss of CRBP1 mRNA, and in vitro treatment with the demethylating agent 5-aza-2'-deoxycytidine reactivated CRBP1 expression. CRBP1 methylation appeared in premalignant lesions and frequently occurred with RARbeta2 hypermethylation in the same tumors. Finally, we observed that a higher dietary retinol intake was associated with reduced frequencies of methylation of both genes. Epigenetic disruption of CRBP1 is a common event in human cancer that may have important implications for cancer prevention and treatment using retinoids.
Hunt LE, Eichenberger MR, Petras R, Galandiuk SUse of a microsatellite marker in predicting dysplasia in ulcerative colitis.
Arch Surg. 2000; 135(5):582-5 [PubMed
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BACKGROUND: Patients with ulcerative colitis (UC) have an increased risk of developing colorectal cancer. The current screening protocol involves an annual colonoscopy and biopsy after the patient has had the disease for 8 years. This, however, does not prevent the development of colorectal cancer.
HYPOTHESIS: A microsatellite marker for IBD1 may identify individuals who are at greater risk of developing dysplasia and therefore colorectal cancer.
DESIGN: Case-control study.
SETTING: Single surgical practice.
PATIENTS AND METHODS: DNA was extracted from peripheral leukocytes of 152 patients: 22 with UC and dysplasia; 48 with UC and no dysplasia; 24 with colorectal cancer; and 58 with noninflammatory bowel disease, nonmalignant gastrointestinal tract disease who were used as control patients. A microsatellite marker for IBD1 (D16S541) was amplified by polymerase chain reaction. Genotypes were identified using autoradiography.
RESULTS: Six alleles and 15 genotypes were identified for marker D 16S541. Genotype CC was found in 33% (8/24) of cancer patients but only 12% (7/58) of controls (chi2 = 5.5; P = .02). Thirty-two percent (7/22) of patients with dysplastic UC also had this genotype, whereas only 8% (4/ 48) of patients with nondysplastic UC had the genotype (chi2 = 4.6; P = .03; vs controls: chi2 = 3.1; P = .08).
CONCLUSIONS: This microsatellite marker for IBD1, when combined with other markers, has the potential to be used as a screening tool for colorectal cancer and dysplasia in patients with UC. Such a marker would be of particular use in improving the sensitivity and specificity of the current screening protocol for dysplasia and colorectal cancer for patients with UC.