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Epigenetic versus genetic alterations in the inactivation of E-cadherin
seminars in CANCER BIOLOGY, Vol. 12, 2002: pp. 373–379 doi:10.1016/S1044–579X(02)00057-3, available online at http://www.idealibrary.com on
Epigenetic versus genetic alterations in the inactivation of E-cadherin Gordon Strathdee
for activation of oncogenes or inactivation of tumour suppressors, leading to the development of tumours.1 However, in recent years it has become increasingly obvious that genetic abnormalities are by no means the only mechanism by which gene expression becomes altered during tumourigenesis. Growing evidence now suggests that epigenetic factors, in particular DNA methylation, play a major role in carcinogenesis and indeed may be as significant as the more widely studied genetic abnormalities.2 DNA methylation is the only commonly occurring modification of human DNA and results from the activity of a family of DNA methyltransferase enzymes that catalyse the addition of a methyl group to cytosine residues at CpG dinucleotides.3 The human genome exhibits a clear depletion of CpG dinucleotides, presumably due to the high rate of deamination of 5-methyl cytosine to thymine.3 However, the genome also contains small stretches, up to a few kilobases in length, that are comparatively rich in CpG dinucleotides, known as CpG islands, which are often found associated with the promoter regions of genes. Unlike the bulk of DNA, where the CpG dinucleotides are highly methylated, the CpG dinucleotides in these islands are usually methylation free in adult tissue and this pattern of DNA methylation is stably inherited from one cell generation to the next.3 The potential functional importance of CpG islands was revealed by studies demonstrating that methylation of CpG islands within gene promoters is associated with transcriptional repression of the associated gene.4 In normal tissue, for the majority of genes associated with CpG islands, the island remains methylation free regardless of whether the gene is expressed or not.3 However, almost all human cancer types appear to show a loss of the normal control of DNA methylation, resulting in both an overall decrease in the genome wide level of methylation, in association with local areas of increased methylation, centring on CpG islands.2 Indeed, recently developed methods that allow large-scale analysis of CpG islands indicate that
Genetic mutation of genes that inhibit the formation of tumours has long been known to be one of the main driving forces in the development of cancer. Inactivation of one such gene, E-cadherin, is thought to be an important step in the development of most, or all, epithelial derived tumour types. Mutations within the E-cadherin gene have been identified as the cause of familial gastric cancer and loss of expression of E-cadherin has been found to be widespread in sporadically occurring epithelial tumours. Despite this, mutations of the E-cadherin gene have been only rarely found in most types of sporadic cancers. However, recent evidence has identified a second mechanism potentially responsible for inactivation of E-cadherin, and other important genes, during tumourigenesis, namely DNA methylation. This review will examine the importance of genetic (mutation) versus epigenetic (DNA methylation) mechanisms in the inactivation of E-cadherin during tumour development and also discuss potential differences in the functional consequences between inactivation by epigenetic or genetic means. Key words: E-cadherin / epigenetics / DNA methylation / cell adhesion / metastasis © 2002 Elsevier Science Ltd. All rights reserved.
Introduction Cancer has long been known to be a genetic disease, with a variety of different genetic abnormalities being associated with all types of cancer. Point mutations, deletions, insertions, amplifications and translocations have all been shown to be responsible From the Cancer Research Campaign Department of Medical Oncology, CRC Beatson Laboratories, Glasgow University, Glasgow G61 1BD, UK. E-mail: [email protected] © 2002 Elsevier Science Ltd. All rights reserved. 1044–579X / 02 / $ – see front matter
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an important role in signal transduction.11 A large number of reports have identified down-regulation of E-cadherin expression in many human carcinomas.8 and indeed E-cadherin’s function is lost during the development of most epithelial cancers (for review, see References 9,12). Loss of E-cadherin function in cancer cells likely plays at least two different roles in tumour development and progression. Most of the studies on E-cadherin and cancer have focussed on its role in suppression of metastasis.12 Loss of E-cadherin expression was demonstrated to be associated with increased invasiveness in many human tumour types.8 Furthermore, re-expression of E-cadherin in cell culture results in reversion of the invasive phenotype.13,14 These results strongly suggest that E-cadherin plays a major role as a suppressor of invasion in human tumours. However, recent studies on familial gastric cancer indicate that E-cadherin can also act at a much earlier stage during tumour development. Mutations of the E-cadherin gene were found in three familial gastric cancer kindreds from New Zealand15 and this observation was confirmed in kindreds of European origin.16 These results demonstrate that mutation of E-cadherin can play a role in susceptibility to initial tumour development in addition to its role as an inhibitor of tumour invasion. This may be due to E-cadherin’s role in signal transduction and in particular due to the release into the cytoplasm, following loss of E-cadherin, of increased amounts of B-catenin, which is known to modulate gene transcription and play a role in the WNT-mediated signalling pathway.17
cancers probably exhibit aberrantly increased methylation of hundreds, or even thousands, of CpG islands within a single tumour.5,6 The overall decreases in genomic DNA methylation observed in many tumour types may play an important role in tumourigenesis,2 however, the majority of studies linking methylation abnormalities to tumour development have focused on the inactivation of tumour suppressor genes due to hypermethylation of their associated CpG islands. Indeed for many genes known to play important roles in preventing tumour development, it has been shown that reduced expression in tumours is frequently associated with increased DNA methylation.2 Aberrant methylation of CpG islands within apparently normal tissue has also been identified and shown to correlate strongly with increasing age.7 It has been suggested that this age-dependent accumulation of methylation abnormalities may be one of the primary reasons for the strong link between the incidence of most cancers and increasing age.7 E-cadherin is a cell adhesion molecule that is essential to maintaining the integrity of cell–cell contact in epithelial cell layers.8 Mutations within the E-cadherin gene have been shown to be associated with familial gastric cancer and loss of expression of E-cadherin in sporadic tumours is thought to be a key step in metastasis.9 However, although a high frequency of E-cadherin mutations have been found in two specific subtypes of cancer, for the majority of epithelial-derived tumours mutations of E-cadherin have rarely, if ever, been reported.10 Consequently, the study of alternative mechanisms for inactivation of E-cadherin, in particular, increased DNA methylation of its promoter, has received much recent attention. This review will compare the role of the more established genetic alterations to the more recently defined role of hypermethylation in the inactivation of E-cadherin and address the possibility that the selection of epigenetic or genetic mechanisms of inactivation may not be random but due to functional differences in the their consequences on E-cadherin function.
Genetic inactivation of E-cadherin The E-cadherin gene can be genetically inactivated by a number of mechanisms. The first hints of a role for E-cadherin in tumour development, particularly in suppression of invasion came from loss of heterozygosity studies on chromosome 16. In 1989, E-cadherin was shown to map to chromosome 16q22.1.18 The following year two reports in hepatocellular carcinoma and breast cancer identified loss of heterozygosity in this region, which was associated with tumours of high metastatic potential.19,20 The first mutations of the E-cadherin gene were reported in two loosely adherent gastric carcinoma cell lines.20 and subsequently mutations were reported in tumour samples in gynaecologic cancers,21 diffuse type gastric carcinomas22 and infiltrative lobular breast cancer.23,24 However,
Role of E-cadherin in normal cells and cancer E-cadherin is a calcium dependent cell adhesion molecule, which is found predominantly in epithelial cells and plays a pivotal role in maintaining tissue integrity.8 In addition, E-cadherin interacts with a number of proteins via its intercellular domain, known as catenins, through which it appears to play 374
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Table 1. Mechanisms of inactivation of E-cadherin in sporadic human tumours Tumour type
Frequency of mutation (%)
Frequency of hypermethylation (%)
Frequency of LOH (%)a
Diffuse gastric cancer Other gastric cancer Lobular breast cancer Other breast cancer Colorectal Bladder Leukaemia Oesophageal Hepatocellular carcinoma Synovial sarcoma Thyroid Uterine Oral SCC Prostate
4122,44–46 022,45,46 3223,25,50,51 025,39,53 054,55 357 – 062 064 2467 468,69 421 072 074,75
7544,47,48 4947,48 7750 4431,39 4655 4333 4859,60 8236,63 4165,66 – 4368,70 2835 3672,73 5476
2449 1049 8125,50,51,52 3839,52,53 1356 1458 261 6862 4664–66 – 769 1571 – 3877,78
Superscripted numbers indicate references. a Loss of heterozygosity.
although down-regulation of E-cadherin expression is commonly seen in many tumour types,8 mutations in E-cadherin were not found to be common and appear to be specific for certain subtypes of cancer (Table 1). In the study of gynaecologic cancer only 4 of 135 carcinomas exhibited detectable mutations and of these only 2 appeared to have loss of the remaining wild type allele.21 In breast cancer, E-cadherin mutations are common in lobular breast cancer, but not in ductal or medullary samples.24,25 Similarly, in gastric cancer mutations are common in diffuse type carcinomas, but not seen in intestinal type gastric cancer (Table 1).22 Mutations of E-cadherin in other types of cancer are rarely observed, although deletion of one E-cadherin allele, as detected by loss of heterozygosity, is more widely seen (Table 1). The specificity of types of cancer affected by mutations of the E-cadherin gene, despite the prevalence of reduced E-cadherin expression in many cancer types, suggests that E-cadherin mutations may be of particular importance in the development of these tumours. In addition, mutations in both diffuse gastric26 and lobular breast cancer27 have been detected early in tumour development suggesting a role in tumour suppression, as opposed to invasion suppression, at least in these two tumour types. Further evidence for this comes from the observations that mutations of E-cadherin have also been observed in several kindreds exhibiting familial gastric cancer.15,16 Furthermore, at least one kindred exhibited both diffuse gastric cancer and early-onset breast cancer.28
Inactivation by hypermethylation In recent years it has become increasingly apparent that increased methylation within the promoter regions of genes plays a key role in the inactivation of many important genes during the development of cancer.2 E-cadherin promoter methylation was first reported by Yoshiura et al.29 in a number of human carcinoma cell lines and was shown to be associated with transcription inactivation of the gene. Furthermore, reversal of promoter methylation, by treatment with 5-azacytidine, resulted in re-activation of E-cadherin expression. Soon afterwards Graff et al.30 reported similar findings in breast and prostate carcinoma cell lines and also in primary breast tumours. Crucially, the authors also demonstrated, by transfection of reporter genes whose expression was driven by the E-cadherin promoter, that E-cadherin negative cell lines still retained the ability to transactivate the E-cadherin promoter. These results confirm that in cancer cell lines E-cadherin transcriptional repression was maintained by promoter hypermethylation. Subsequently, numerous reports of E-cadherin promoter methylation, associated with reduced E-cadherin expression, have been presented (Table 1) and methylation is frequently associated with disease progression and metastasis.31 Unlike mutational inactivation, which is frequent in only two specific tumour types, hypermethylation of E-cadherin is seen in a wide range of human tumours (Table 1). Interestingly, hypermethylation has also been shown to play a role in familial gastric 375
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mutations are seen early in tumour development,26,27 frequently in association with deletion of the remaining normal allele.10 Furthermore, the spectrum of tumours caused by germline mutations of E-cadherin closely mirrors that of sporadic tumours with E-cadherin mutations, suggesting E-cadherin only has a role in early tumour development of these particular tumour types.15,16,28 In contrast, in the majority of epithelial-derived cancers, E-cadherin expression is almost always lost in association with hypermethylation (Table 1) and its loss is probably primarily involved in metastasis, as opposed to initial tumour development. At this stage their may be a balance between selection for reduced E-cadherin, allowing tumour cells to invade into areas with increased access to nutrients, and selection against E-cadherin loss, due to its role in preventing apoptosis.37 This may favour epigenetic mechanisms which can lead to different levels of down-regulation of expression depending on methylation density.38 Cheng et al.39 demonstrated, in ductal breast cancer, that LOH at the E-cadherin locus and hypermethylation of the promoter were both commonly observed in this tumour population. However, contrary to the predictions of the Knudson’s standard two-hit model for tumour suppressor inactivation, which would have predicted that hypermethylation should exhibit a positive correlation with LOH, to give the two hits, LOH and methylation were actually shown to be inversely correlated. Based on this and other observations the authors propose that there is actually selection for retention of at least some limited E-cadherin expression or for mechanisms of inactivation, such as promoter hypermethylation, which are more readily reversible than genetic mutations. And, indeed, E-cadherin expression is frequently restored in cells from metastatic lesions.40 Further support for such a model comes from detailed analysis of the E-cadherin promoter performed by Graff et al.41 Using breast and prostate cancer cell lines the authors demonstrated that the E-cadherin promoter showed a heterogeneous pattern of methylation and that the distribution of methylation, and consequently the level of E-cadherin expression, could be greatly influenced by the microenvironment of the cells. Thus, cells that had invaded through a basement membrane exhibited increased methylation of the promoter and reduced E-cadherin expression. On the other hand, when the same cells were grown as spheroids, which favours high expression of E-cadherin, reduced methylation of the promoter and increased expression were observed. Even MCF7 cells, which display complete
cancer, acting as the second hit in inactivation of E-cadherin.32 E-cadherin may also be part of the subset of genes which are susceptible to age-related methylation.7 Bornman et al.33 showed that the E-cadherin gene was aberrantly methylated in histologically normal bladder samples from three elderly individuals. However, such age-related methylation of E-cadherin may not be widespread as a number of other studies failed to detect evidence of E-cadherin methylation in normal tissue samples.34–36
Epigenetic versus genetic inactivation—which to choose? E-cadherin, similar to a growing list of important genes in cancer, is clearly targeted by both epigenetic and genetic mechanisms of inactivation during tumour development and progression. However, it is less clear what determines which of the two mechanisms will inactivate a particular gene in a particular tumour and whether there are differences in the functional consequences between inactivation by one mechanisms or the other. In the case of E-cadherin, the differences in the balance between genetic and epigenetic events in different tumour types (i.e. the high frequency of mutations in two tumour types, but nearly exclusive epigenetic inactivation in most other cancers (Table 1)) suggests that such differences in functional consequences may well exist. Alternatively, abnormalities of DNA methylation could just be rare in diffuse gastric and lobular breast cancer leading to a stronger selective pressure for mutational inactivation. However, several reports indicate that methylation of the E-cadherin locus is in fact very common in both tumour types. If methylation abnormalities are still common in lobular breast and diffuse gastric cancer, why then are mutations seen so commonly in these tumour types, but not others. A potential explanation could lie in the two apparent roles of E-cadherin, in tumour development or in tumour metastasis. Mutations within the E-cadherin gene may be predominantly found in tumours where complete loss of E-cadherin function plays a vital role in early tumourigenesis, as opposed to primarily in metastasis, and thus there is a strong selective pressure for complete inactivation of E-cadherin early in tumour development. This would be supported by the findings that almost all mutations of E-cadherin identified in diffuse gastric and lobular breast cancer are predicted to result in complete inactivation of normal protein function10 and that 376
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methylation of the promoter and complete loss of E-cadherin expression if grown as a monolayer, when grown as spheroids, still exhibited reduced promoter methylation and re-expression of E-cadherin. This last result emphasises the comparatively unstable nature of epigenetic alterations, which may be crucial for allowing tumour cells to alter E-cadherin expression at different stages of invasion and metastasis. However, a second potential model for the high rate of epigenetic inactivation of E-cadherin could be proposed involving the transcriptional repressor Snail. It is important to note that although hypermethylation of gene promoters has been extensively associated with maintenance of transcriptional inactivation of genes in cancer,2 it is still unknown whether methylation itself induces loss of expression or if it occurs secondary to another mechanism which is responsible for an initial down-regulation of expression. In the report referred to above, Cheng et al.39 also investigated the level of expression of the transcriptional regulator Snail, which has been demonstrated to negatively regulate E-cadherin expression,42,43 and found that increased Snail expression was strongly correlated with reduced E-cadherin expression. Importantly though, increased Snail expression showed no correlation with LOH, but did show a clear positive correlation with promoter hypermethylation. This correlation could be explained if the frequency with which methylation targets the E-cadherin promoter increased when the promoter activity is down-regulated by other mechanisms, such as increased Snail expression. Therefore, if down-regulation of E-cadherin, due to increased Snail expression or alterations in other transcriptional regulators, is commonly selected for during tumour development this could shift the balance of epigenetic and genetic inactivation in favour of epigenetic inactivation of E-cadherin.
of E-cadherin being seen frequently in most tumour types, but mutations only frequent in a small number of specific subtypes. These results are compatible with the idea that epigenetic and genetic inactivation of genes could potentially have different roles in tumour development. In the specific case of E-cadherin, the comparatively unstable nature of epigenetic changes may be vital in allowing invading tumour cells to alter E-cadherin levels in response to different microenvironments. However, in cancers where E-cadherin is of greater importance in initial tumourigenesis, complete loss of E-cadherin may be required, and thus permanent inactivation by genetic mutation may be more strongly favoured. However, although the comparatively reversible nature of epigenetic lesions may be a selective advantage at sites of distant metastases, the prevalence of epigenetic lesions in primary tumours indicates that such selective pressures would have to act at the site of the primary tumour and it is not clear what the selective pressures would be that favour epigenetic as opposed to genetic inactivation in the early stages of metastasis. Therefore, further work will be required defining the biological effects of different levels of E-cadherin expression in tumour cell populations. In addition, it will also be of interest to determine whether other genes exhibit similar differences in the functional consequences of epigenetic versus genetic inactivation during tumourigenesis.
Acknowledgements The author would like to thank Prof. Robert Brown for critical reading of the manuscript. This work was supported by Cancer Research UK.
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In most epithelial tumour types, E-cadherin levels are likely to be an important factor in determining metastatic potential, however, the association between familial gastric cancer and germline E-cadherin mutations strongly suggests that loss of E-cadherin can also be important in the early stages of tumour development. Like many other genes that inhibit tumour development and progression, E-cadherin can be targeted by both genetic and epigenetic means. The distribution of epigenetic versus genetic inactivation though is clearly not random, with hypermethylation 377
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46. Machado JC, Soares P, Carneiro F, Rocha A, Beck S, Blin N, Berx G, Sobrihho-Simoes M (1999) E-cadherin gene mutations provide a genetic basis for the phenotypic divergence of mixed gastric carcinomas. Lab Invest 79:459–465 47. Leung WK et al. (2001) Concurrent hypermethylation of multiple tumour-related genes in gastric carcinoma and adjacent normal tissues. Cancer 91:2294–2301 48. Tamura G et al. (2000) E-cadherin gene promoter hypermethylation in primary human gastric cancer. J Natl Cancer Inst 92:569– 573 49. Kimura T, Sugihara H, Misufuji S, Kodama T, Hattori T (1998) Microsatellite analysis and DNA ploidy pattern in signet-ring cell carcinomas and well differentiated adenocarcinomas of the stomach. Cancer J 11:298–305 50. Droufakou S, Deshmane V, Roylance R, Hanby A, Tomlinson I, Hart IR (2001) Multiple ways of silencing E-cadherin gene expression in lobular carcinoma of the breast. Int J Cancer 92:404– 408 51. Huiping C, Sigurgeirsdottir JR, Jonasson JG, Eiriksdottir G, Johannsdottir JT, Egilsson V, Ingvarsson S (1999) Chromosome alterations and E-cadherin gene mutations in human lobular breast cancer. Br J Cancer 81:1103–1110 52. Asgeirsson KS, Jonasson JG, Tryggvadottir L, Olafsdottir K, Sigurgeirsdottir JR, Ingvarsson S, Ogmundsdottir HM (2000) Altered expression of E-cadherin in breast cancer: patterns, mechanisms and clinical significance. Eur J Cancer 36:1098– 1106 53. Kashiwaba M, Tamura G, Suzuki Y, Maesawa C, Ogasawara S, Sakata K, Satodate R (1995) Epithelial-cadherin gene is not mutated in ductal carcinomas of the breast. Jpn J Cancer Res 86:1054–1059 54. Schumacher C et al. (1999) Loss of immunohistochemical E-cadherin expression in colon cancer is not due to structural gene alterations. Virchows Archiv 434:489–495 55. Wheeler JM, Kim HC, Efstathiou JA, Ilyas M, Mortensen MJ, Bodmer WF (2001) Hypermethylation of the promoter region of the E-cadherin gene (CDH1) in sporadic and ulcerative colitis associated colorectal cancer. Gut 48:367–371 56. Tomlinson I, Ilyas M, Johnson V, Davies A, Clark G, Talbot I, Bodmer W (1998) A comparison of the genetic pathways involved in the pathogenesis of three types of colorectal cancer. J Pathol 184:148–152 57. Taddei I, Piazzini M, Bartoletti R, Dal Canto M, Sardi I (2000) Molecular alterations of E-cadherin gene: possible role in human bladder carcinogenesis. Int J Mol Med 6:201–208 58. Yoon DS et al. (2001) Genetic mapping and DNA sequencebased analysis of deleted regions on chromosome 16 involved in progression of bladder cancer from occult preneoplastic conditions to invasive disease. Oncogenesis 20:5005–5014 59. Corn PG, Smith BD, Ruckdeschel DS, Douglas D, Baylin SB, Herman JG (2000) E-cadherin expression is silenced by 5 CpG island methylation in acute leukemia. Clin Cancer Res 6:4243– 4248 60. Melki JRPCV, Brown RD, Clark SJ (2000) Hypermethylation of E-cadherin in leukemia. Blood 95:3208–3213 61. Sweetster DA et al. (2001) Loss of heterozygosity in childhood de novo acute myelogenous leukemia. Blood 98:1188–1194 62. Wijnhoven BPL, de Both NJ, van Dekken H, Tilanus HW, Dinjens WNM (1999) E-cadherin gene mutations are rare in adenocarcinoma of the oesophagus. Br J Cancer 80:1652–1657
63. Si HX, Tsao SW, Lam KY, Srivastava G, Liu Y, Wong YC, Shen ZY, Cheung AL (2001) E-cadherin expression is commonly down-regulated by CpG hypermethylation in esophageal carcinoma cells. Cancer Lett 173:71–78 64. Wang G et al. (2000) Genetic aberration in primary hepatocellualr carcinoma: correlation between p53 gene mutation and loss-of-heterozygosity on chromosome 16q21–q23 and p21–p23. Cell Res 10:311–323 65. Matsumura T, Makino R, Mitamura K (2001) Frequent downregulation of E-cadherin by genetic and epigenetic changes in the malignant progression of hepatocellular carcinomas. Clin Cancer Res 7:594–599 66. Kanai YSU, Tsuda H, Sakamoto M, Hirohashi S (2000) Aberrant DNA methylation precedes loss of heterozygosity on chromosome 16 in chronic hepatitis and liver cirrhosis. Cancer Lett 148:73–80 67. Saito T et al. (2001) E-cadherin gene mutations frequently occur in synovial sarcoma as a determinant of hitological features. Am J Pathol 159:2117–2124 68. Rocha AS, Soares P, Seruca R, Maximo V, Matias-Guiu X, Cameselle-Teijeiro J, Sobrihho-Simoes M (2001) Abnormalities of the E-cadherin/catenin complex in classical papillary thyroid carcinoma and in its diffuse sclerosing variant. J Pathol 194:358– 366 69. Soares P, Berx G, van Roy F, Sobrinho-Simoes M (1997) E-cadherin gene alterations are rare events in thyroid tumours. Int J Cancer 70:32–38 70. Graff JR et al. (1998) Distinct patterns of E-cadherin CpG island methylstion in papillary, follicular, Hurtlhe’s cell, and poorly differentiated human thyroid carcinoma. Cancer Res 58:2063– 2066 71. Krul EJT, Kersemaekers AJF, Zomerdijik-Nooyen YA, Cornelisse CJ, Peters LAW, Fleuren GJ (1999) Different profiles of allelic loss in cervical carcinoma cases in Surinam and The Netherlands. Cancer 86:997–1004 72. Saito YHT, Uzawa K, Tanzawa H, Sato K (1998) Reduced expression of E-cadherin in oral squamous cell carcinoma: relationship with DNA methylation of 5 CpG island. Int J Oncol 12:293–298 73. Nakayama S, Sasaki A, Mese H, Alcalde RE, Tsuki T, Matsumura T (2001) The E-cadherin gene is silenced by CpG methylation in human oral squamous cell carcinomas. Br J Cancer 93:667–673 74. Li CD et al. (1999) Distinct deleted regions on chromosome segment 16q23–q24 associated with metastasis in prostate cancer. Genes Chromosomes & Cancer 24:175–182 75. Suzuki H, Komiya A, Emi M, Kuramochi H, Shiraishi T, Yatani R, Shimazaki J (1996) Three distinct commonly deleted regions of chromosome arm 16q in human primary and metastatic prostate carcinomas. Gen Chrom Cancer 17:225–233 76. Li LC, Zhao H, Nakajima K, Oh BR, Filho LA, Carroll P, Dahiya R (2001) Methylation of the E-cadherin gene promoter correlates with progression of prostate cancer. J Urol 166:705–709 77. Latil A, Cussenot O, Fournier G, Driouch K, Lidereau R (1997) Loss of heterozygosity of chromosome 16q in prostate adenocarcinoma: identification of three independent regions. Cancer Res 57:1058–1062 78. Elo JP, Harkonen P, Kyllonen AP, Lukkarinen O, Poutanen M, Vihko R, Vihko P (1997) Loss of heterozygosity at 16q24–q24.2 is significantly associated with metastatic and aggressive behaviour of prostate cancer. Cancer Res 57:3356–3359
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for activation of oncogenes or inactivation of tumour suppressors, leading to the development of tumours.1 However, in recent years it has become increasingly obvious that genetic abnormalities are by no means the only mechanism by which gene expression becomes altered during tumourigenesis. Growing evidence now suggests that epigenetic factors, in particular DNA methylation, play a major role in carcinogenesis and indeed may be as significant as the more widely studied genetic abnormalities.2 DNA methylation is the only commonly occurring modification of human DNA and results from the activity of a family of DNA methyltransferase enzymes that catalyse the addition of a methyl group to cytosine residues at CpG dinucleotides.3 The human genome exhibits a clear depletion of CpG dinucleotides, presumably due to the high rate of deamination of 5-methyl cytosine to thymine.3 However, the genome also contains small stretches, up to a few kilobases in length, that are comparatively rich in CpG dinucleotides, known as CpG islands, which are often found associated with the promoter regions of genes. Unlike the bulk of DNA, where the CpG dinucleotides are highly methylated, the CpG dinucleotides in these islands are usually methylation free in adult tissue and this pattern of DNA methylation is stably inherited from one cell generation to the next.3 The potential functional importance of CpG islands was revealed by studies demonstrating that methylation of CpG islands within gene promoters is associated with transcriptional repression of the associated gene.4 In normal tissue, for the majority of genes associated with CpG islands, the island remains methylation free regardless of whether the gene is expressed or not.3 However, almost all human cancer types appear to show a loss of the normal control of DNA methylation, resulting in both an overall decrease in the genome wide level of methylation, in association with local areas of increased methylation, centring on CpG islands.2 Indeed, recently developed methods that allow large-scale analysis of CpG islands indicate that
Genetic mutation of genes that inhibit the formation of tumours has long been known to be one of the main driving forces in the development of cancer. Inactivation of one such gene, E-cadherin, is thought to be an important step in the development of most, or all, epithelial derived tumour types. Mutations within the E-cadherin gene have been identified as the cause of familial gastric cancer and loss of expression of E-cadherin has been found to be widespread in sporadically occurring epithelial tumours. Despite this, mutations of the E-cadherin gene have been only rarely found in most types of sporadic cancers. However, recent evidence has identified a second mechanism potentially responsible for inactivation of E-cadherin, and other important genes, during tumourigenesis, namely DNA methylation. This review will examine the importance of genetic (mutation) versus epigenetic (DNA methylation) mechanisms in the inactivation of E-cadherin during tumour development and also discuss potential differences in the functional consequences between inactivation by epigenetic or genetic means. Key words: E-cadherin / epigenetics / DNA methylation / cell adhesion / metastasis © 2002 Elsevier Science Ltd. All rights reserved.
Introduction Cancer has long been known to be a genetic disease, with a variety of different genetic abnormalities being associated with all types of cancer. Point mutations, deletions, insertions, amplifications and translocations have all been shown to be responsible From the Cancer Research Campaign Department of Medical Oncology, CRC Beatson Laboratories, Glasgow University, Glasgow G61 1BD, UK. E-mail: [email protected] © 2002 Elsevier Science Ltd. All rights reserved. 1044–579X / 02 / $ – see front matter
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an important role in signal transduction.11 A large number of reports have identified down-regulation of E-cadherin expression in many human carcinomas.8 and indeed E-cadherin’s function is lost during the development of most epithelial cancers (for review, see References 9,12). Loss of E-cadherin function in cancer cells likely plays at least two different roles in tumour development and progression. Most of the studies on E-cadherin and cancer have focussed on its role in suppression of metastasis.12 Loss of E-cadherin expression was demonstrated to be associated with increased invasiveness in many human tumour types.8 Furthermore, re-expression of E-cadherin in cell culture results in reversion of the invasive phenotype.13,14 These results strongly suggest that E-cadherin plays a major role as a suppressor of invasion in human tumours. However, recent studies on familial gastric cancer indicate that E-cadherin can also act at a much earlier stage during tumour development. Mutations of the E-cadherin gene were found in three familial gastric cancer kindreds from New Zealand15 and this observation was confirmed in kindreds of European origin.16 These results demonstrate that mutation of E-cadherin can play a role in susceptibility to initial tumour development in addition to its role as an inhibitor of tumour invasion. This may be due to E-cadherin’s role in signal transduction and in particular due to the release into the cytoplasm, following loss of E-cadherin, of increased amounts of B-catenin, which is known to modulate gene transcription and play a role in the WNT-mediated signalling pathway.17
cancers probably exhibit aberrantly increased methylation of hundreds, or even thousands, of CpG islands within a single tumour.5,6 The overall decreases in genomic DNA methylation observed in many tumour types may play an important role in tumourigenesis,2 however, the majority of studies linking methylation abnormalities to tumour development have focused on the inactivation of tumour suppressor genes due to hypermethylation of their associated CpG islands. Indeed for many genes known to play important roles in preventing tumour development, it has been shown that reduced expression in tumours is frequently associated with increased DNA methylation.2 Aberrant methylation of CpG islands within apparently normal tissue has also been identified and shown to correlate strongly with increasing age.7 It has been suggested that this age-dependent accumulation of methylation abnormalities may be one of the primary reasons for the strong link between the incidence of most cancers and increasing age.7 E-cadherin is a cell adhesion molecule that is essential to maintaining the integrity of cell–cell contact in epithelial cell layers.8 Mutations within the E-cadherin gene have been shown to be associated with familial gastric cancer and loss of expression of E-cadherin in sporadic tumours is thought to be a key step in metastasis.9 However, although a high frequency of E-cadherin mutations have been found in two specific subtypes of cancer, for the majority of epithelial-derived tumours mutations of E-cadherin have rarely, if ever, been reported.10 Consequently, the study of alternative mechanisms for inactivation of E-cadherin, in particular, increased DNA methylation of its promoter, has received much recent attention. This review will compare the role of the more established genetic alterations to the more recently defined role of hypermethylation in the inactivation of E-cadherin and address the possibility that the selection of epigenetic or genetic mechanisms of inactivation may not be random but due to functional differences in the their consequences on E-cadherin function.
Genetic inactivation of E-cadherin The E-cadherin gene can be genetically inactivated by a number of mechanisms. The first hints of a role for E-cadherin in tumour development, particularly in suppression of invasion came from loss of heterozygosity studies on chromosome 16. In 1989, E-cadherin was shown to map to chromosome 16q22.1.18 The following year two reports in hepatocellular carcinoma and breast cancer identified loss of heterozygosity in this region, which was associated with tumours of high metastatic potential.19,20 The first mutations of the E-cadherin gene were reported in two loosely adherent gastric carcinoma cell lines.20 and subsequently mutations were reported in tumour samples in gynaecologic cancers,21 diffuse type gastric carcinomas22 and infiltrative lobular breast cancer.23,24 However,
Role of E-cadherin in normal cells and cancer E-cadherin is a calcium dependent cell adhesion molecule, which is found predominantly in epithelial cells and plays a pivotal role in maintaining tissue integrity.8 In addition, E-cadherin interacts with a number of proteins via its intercellular domain, known as catenins, through which it appears to play 374
Epigenetic versus genetic inactivation of E-cadherin
Table 1. Mechanisms of inactivation of E-cadherin in sporadic human tumours Tumour type
Frequency of mutation (%)
Frequency of hypermethylation (%)
Frequency of LOH (%)a
Diffuse gastric cancer Other gastric cancer Lobular breast cancer Other breast cancer Colorectal Bladder Leukaemia Oesophageal Hepatocellular carcinoma Synovial sarcoma Thyroid Uterine Oral SCC Prostate
4122,44–46 022,45,46 3223,25,50,51 025,39,53 054,55 357 – 062 064 2467 468,69 421 072 074,75
7544,47,48 4947,48 7750 4431,39 4655 4333 4859,60 8236,63 4165,66 – 4368,70 2835 3672,73 5476
2449 1049 8125,50,51,52 3839,52,53 1356 1458 261 6862 4664–66 – 769 1571 – 3877,78
Superscripted numbers indicate references. a Loss of heterozygosity.
although down-regulation of E-cadherin expression is commonly seen in many tumour types,8 mutations in E-cadherin were not found to be common and appear to be specific for certain subtypes of cancer (Table 1). In the study of gynaecologic cancer only 4 of 135 carcinomas exhibited detectable mutations and of these only 2 appeared to have loss of the remaining wild type allele.21 In breast cancer, E-cadherin mutations are common in lobular breast cancer, but not in ductal or medullary samples.24,25 Similarly, in gastric cancer mutations are common in diffuse type carcinomas, but not seen in intestinal type gastric cancer (Table 1).22 Mutations of E-cadherin in other types of cancer are rarely observed, although deletion of one E-cadherin allele, as detected by loss of heterozygosity, is more widely seen (Table 1). The specificity of types of cancer affected by mutations of the E-cadherin gene, despite the prevalence of reduced E-cadherin expression in many cancer types, suggests that E-cadherin mutations may be of particular importance in the development of these tumours. In addition, mutations in both diffuse gastric26 and lobular breast cancer27 have been detected early in tumour development suggesting a role in tumour suppression, as opposed to invasion suppression, at least in these two tumour types. Further evidence for this comes from the observations that mutations of E-cadherin have also been observed in several kindreds exhibiting familial gastric cancer.15,16 Furthermore, at least one kindred exhibited both diffuse gastric cancer and early-onset breast cancer.28
Inactivation by hypermethylation In recent years it has become increasingly apparent that increased methylation within the promoter regions of genes plays a key role in the inactivation of many important genes during the development of cancer.2 E-cadherin promoter methylation was first reported by Yoshiura et al.29 in a number of human carcinoma cell lines and was shown to be associated with transcription inactivation of the gene. Furthermore, reversal of promoter methylation, by treatment with 5-azacytidine, resulted in re-activation of E-cadherin expression. Soon afterwards Graff et al.30 reported similar findings in breast and prostate carcinoma cell lines and also in primary breast tumours. Crucially, the authors also demonstrated, by transfection of reporter genes whose expression was driven by the E-cadherin promoter, that E-cadherin negative cell lines still retained the ability to transactivate the E-cadherin promoter. These results confirm that in cancer cell lines E-cadherin transcriptional repression was maintained by promoter hypermethylation. Subsequently, numerous reports of E-cadherin promoter methylation, associated with reduced E-cadherin expression, have been presented (Table 1) and methylation is frequently associated with disease progression and metastasis.31 Unlike mutational inactivation, which is frequent in only two specific tumour types, hypermethylation of E-cadherin is seen in a wide range of human tumours (Table 1). Interestingly, hypermethylation has also been shown to play a role in familial gastric 375
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mutations are seen early in tumour development,26,27 frequently in association with deletion of the remaining normal allele.10 Furthermore, the spectrum of tumours caused by germline mutations of E-cadherin closely mirrors that of sporadic tumours with E-cadherin mutations, suggesting E-cadherin only has a role in early tumour development of these particular tumour types.15,16,28 In contrast, in the majority of epithelial-derived cancers, E-cadherin expression is almost always lost in association with hypermethylation (Table 1) and its loss is probably primarily involved in metastasis, as opposed to initial tumour development. At this stage their may be a balance between selection for reduced E-cadherin, allowing tumour cells to invade into areas with increased access to nutrients, and selection against E-cadherin loss, due to its role in preventing apoptosis.37 This may favour epigenetic mechanisms which can lead to different levels of down-regulation of expression depending on methylation density.38 Cheng et al.39 demonstrated, in ductal breast cancer, that LOH at the E-cadherin locus and hypermethylation of the promoter were both commonly observed in this tumour population. However, contrary to the predictions of the Knudson’s standard two-hit model for tumour suppressor inactivation, which would have predicted that hypermethylation should exhibit a positive correlation with LOH, to give the two hits, LOH and methylation were actually shown to be inversely correlated. Based on this and other observations the authors propose that there is actually selection for retention of at least some limited E-cadherin expression or for mechanisms of inactivation, such as promoter hypermethylation, which are more readily reversible than genetic mutations. And, indeed, E-cadherin expression is frequently restored in cells from metastatic lesions.40 Further support for such a model comes from detailed analysis of the E-cadherin promoter performed by Graff et al.41 Using breast and prostate cancer cell lines the authors demonstrated that the E-cadherin promoter showed a heterogeneous pattern of methylation and that the distribution of methylation, and consequently the level of E-cadherin expression, could be greatly influenced by the microenvironment of the cells. Thus, cells that had invaded through a basement membrane exhibited increased methylation of the promoter and reduced E-cadherin expression. On the other hand, when the same cells were grown as spheroids, which favours high expression of E-cadherin, reduced methylation of the promoter and increased expression were observed. Even MCF7 cells, which display complete
cancer, acting as the second hit in inactivation of E-cadherin.32 E-cadherin may also be part of the subset of genes which are susceptible to age-related methylation.7 Bornman et al.33 showed that the E-cadherin gene was aberrantly methylated in histologically normal bladder samples from three elderly individuals. However, such age-related methylation of E-cadherin may not be widespread as a number of other studies failed to detect evidence of E-cadherin methylation in normal tissue samples.34–36
Epigenetic versus genetic inactivation—which to choose? E-cadherin, similar to a growing list of important genes in cancer, is clearly targeted by both epigenetic and genetic mechanisms of inactivation during tumour development and progression. However, it is less clear what determines which of the two mechanisms will inactivate a particular gene in a particular tumour and whether there are differences in the functional consequences between inactivation by one mechanisms or the other. In the case of E-cadherin, the differences in the balance between genetic and epigenetic events in different tumour types (i.e. the high frequency of mutations in two tumour types, but nearly exclusive epigenetic inactivation in most other cancers (Table 1)) suggests that such differences in functional consequences may well exist. Alternatively, abnormalities of DNA methylation could just be rare in diffuse gastric and lobular breast cancer leading to a stronger selective pressure for mutational inactivation. However, several reports indicate that methylation of the E-cadherin locus is in fact very common in both tumour types. If methylation abnormalities are still common in lobular breast and diffuse gastric cancer, why then are mutations seen so commonly in these tumour types, but not others. A potential explanation could lie in the two apparent roles of E-cadherin, in tumour development or in tumour metastasis. Mutations within the E-cadherin gene may be predominantly found in tumours where complete loss of E-cadherin function plays a vital role in early tumourigenesis, as opposed to primarily in metastasis, and thus there is a strong selective pressure for complete inactivation of E-cadherin early in tumour development. This would be supported by the findings that almost all mutations of E-cadherin identified in diffuse gastric and lobular breast cancer are predicted to result in complete inactivation of normal protein function10 and that 376
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methylation of the promoter and complete loss of E-cadherin expression if grown as a monolayer, when grown as spheroids, still exhibited reduced promoter methylation and re-expression of E-cadherin. This last result emphasises the comparatively unstable nature of epigenetic alterations, which may be crucial for allowing tumour cells to alter E-cadherin expression at different stages of invasion and metastasis. However, a second potential model for the high rate of epigenetic inactivation of E-cadherin could be proposed involving the transcriptional repressor Snail. It is important to note that although hypermethylation of gene promoters has been extensively associated with maintenance of transcriptional inactivation of genes in cancer,2 it is still unknown whether methylation itself induces loss of expression or if it occurs secondary to another mechanism which is responsible for an initial down-regulation of expression. In the report referred to above, Cheng et al.39 also investigated the level of expression of the transcriptional regulator Snail, which has been demonstrated to negatively regulate E-cadherin expression,42,43 and found that increased Snail expression was strongly correlated with reduced E-cadherin expression. Importantly though, increased Snail expression showed no correlation with LOH, but did show a clear positive correlation with promoter hypermethylation. This correlation could be explained if the frequency with which methylation targets the E-cadherin promoter increased when the promoter activity is down-regulated by other mechanisms, such as increased Snail expression. Therefore, if down-regulation of E-cadherin, due to increased Snail expression or alterations in other transcriptional regulators, is commonly selected for during tumour development this could shift the balance of epigenetic and genetic inactivation in favour of epigenetic inactivation of E-cadherin.
of E-cadherin being seen frequently in most tumour types, but mutations only frequent in a small number of specific subtypes. These results are compatible with the idea that epigenetic and genetic inactivation of genes could potentially have different roles in tumour development. In the specific case of E-cadherin, the comparatively unstable nature of epigenetic changes may be vital in allowing invading tumour cells to alter E-cadherin levels in response to different microenvironments. However, in cancers where E-cadherin is of greater importance in initial tumourigenesis, complete loss of E-cadherin may be required, and thus permanent inactivation by genetic mutation may be more strongly favoured. However, although the comparatively reversible nature of epigenetic lesions may be a selective advantage at sites of distant metastases, the prevalence of epigenetic lesions in primary tumours indicates that such selective pressures would have to act at the site of the primary tumour and it is not clear what the selective pressures would be that favour epigenetic as opposed to genetic inactivation in the early stages of metastasis. Therefore, further work will be required defining the biological effects of different levels of E-cadherin expression in tumour cell populations. In addition, it will also be of interest to determine whether other genes exhibit similar differences in the functional consequences of epigenetic versus genetic inactivation during tumourigenesis.
Acknowledgements The author would like to thank Prof. Robert Brown for critical reading of the manuscript. This work was supported by Cancer Research UK.
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In most epithelial tumour types, E-cadherin levels are likely to be an important factor in determining metastatic potential, however, the association between familial gastric cancer and germline E-cadherin mutations strongly suggests that loss of E-cadherin can also be important in the early stages of tumour development. Like many other genes that inhibit tumour development and progression, E-cadherin can be targeted by both genetic and epigenetic means. The distribution of epigenetic versus genetic inactivation though is clearly not random, with hypermethylation 377
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