Manel Esteller

Inactivation of the DNA-Repair Gene MGMT and the Clinical Response of Gliomas to Alkylating Agents

BACKGROUND The DNA-repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) inhibits the killing of tumor cells by alkylating agents. MGMT activity is controlled by a promoter; methylation of the promoter silences the gene in cancer, and the cells no longer produce MGMT. We examined gliomas to determine whether methylation of the MGMT promoter is related to the responsiveness of the tumor to alkylating agents. METHODS We analyzed the MGMT promoter in tumor DNA by a methylation-specific polymerase-chain-reaction assay. The gliomas were obtained from patients who had been treated with carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea, or BCNU). The molecular data were correlated with the clinical outcome. RESULTS The MGMT promoter was methylated in gliomas from 19 of 47 patients (40 percent). This finding was associated with regression of the tumor and prolonged overall and disease-free survival. It was an independent and stronger prognostic factor than age, stage, tumor grade, or performance status. CONCLUSIONS Methylation of the MGMT promoter in gliomas is a useful predictor of the responsiveness of the tumors to alkylating agents.

Gene-expression profiles in hereditary breast cancer.

BACKGROUND Many cases of hereditary breast cancer are due to mutations in either the BRCA1 or the BRCA2 gene. The histopathological changes in these cancers are often characteristic of the mutant gene. We hypothesized that the genes expressed by these two types of tumors are also distinctive, perhaps allowing us to identify cases of hereditary breast cancer on the basis of gene-expression profiles. METHODS RNA from samples of primary tumor from seven carriers of the BRCA1 mutation, seven carriers of the BRCA2 mutation, and seven patients with sporadic cases of breast cancer was compared with a microarray of 6512 complementary DNA clones of 5361 genes. Statistical analyses were used to identify a set of genes that could distinguish the BRCA1 genotype from the BRCA2 genotype. RESULTS Permutation analysis of multivariate classification functions established that the gene-expression profiles of tumors with BRCA1 mutations, tumors with BRCA2 mutations, and sporadic tumors differed significantly from each other. An analysis of variance between the levels of gene expression and the genotype of the samples identified 176 genes that were differentially expressed in tumors with BRCA1 mutations and tumors with BRCA2 mutations. Given the known properties of some of the genes in this panel, our findings indicate that there are functional differences between breast tumors with BRCA1 mutations and those with BRCA2 mutations. CONCLUSIONS Significantly different groups of genes are expressed by breast cancers with BRCA1 mutations and breast cancers with BRCA2 mutations. Our results suggest that a heritable mutation influences the gene-expression profile of the cancer.

Inactivation of the apoptosis effector Apaf-1 in malignant melanoma

Metastatic melanoma is a deadly cancer that fails to respond to conventional chemotherapy and is poorly understood at the molecular level. p53 mutations often occur in aggressive and chemoresistant cancers but are rarely observed in melanoma. Here we show that metastatic melanomas often lose Apaf-1, a cell-death effector that acts with cytochrome c and caspase-9 to mediate p53-dependent apoptosis. Loss of Apaf-1 expression is accompanied by allelic loss in metastatic melanomas, but can be recovered in melanoma cell lines by treatment with the methylation inhibitor 5-aza-2′-deoxycytidine (5aza2dC). Apaf-1-negative melanomas are invariably chemoresistant and are unable to execute a typical apoptotic programme in response to p53 activation. Restoring physiological levels of Apaf-1 through gene transfer or 5aza2dC treatment markedly enhances chemosensitivity and rescues the apoptotic defects associated with Apaf-1 loss. We conclude that Apaf-1 is inactivated in metastatic melanomas, which leads to defects in the execution of apoptotic cell death. Apaf-1 loss may contribute to the low frequency of p53 mutations observed in this highly chemoresistant tumour type.

Cancer as an epigenetic disease: DNA methylation and chromatin alterations in human tumours

Cancer is an epigenetic disease at the same level that it can be considered a genetic disease. In fact, epigenetic changes, particularly DNA methylation, are susceptible to change and are excellent candidates to explain how certain environmental factors may increase the risk of cancer. The delicate organization of methylation and chromatin states that regulates the normal cellular homeostasis of gene expression patterns becomes unrecognizable in the cancer cell. The genome of the transformed cell undergoes simultaneously a global genomic hypomethylation and a dense hypermethylation of the CpG islands associated with gene regulatory regions. These dramatic changes may lead to chromosomal instability, activation of endogenous parasitic sequences, loss of imprinting, illegitimate expression, aneuploidy, and mutations, and may contribute to the transcriptional silencing of tumour suppressor genes. The hypermethylation‐associated inactivation affects virtually all of the pathways in the cellular network, such as DNA repair (hMLH1, BRCA1, MGMT, …︁), the cell cycle (p16INK4a, p14ARF, p15INK4b, …︁), and apoptosis (DAPK, APAF‐1, …︁). The aberrant CpG island methylation can also be used as a biomarker of malignant cells and as a predictor of their behaviour, and may constitute a good target for future therapies. Copyright

MLH1 promoter hypermethylation is associated with the microsatellite instability phenotype in sporadic endometrial carcinomas

Microsatellite instability (MSI) has been detected in endometrial carcinomas occurring in women affected by hereditary nonpolyposis colorectal carcinoma (HNPCC) as well as in 20% of presumably sporadic endometrial tumors. While the MSI+ phenotype observed in endometrial tumors from HNPCC patients is attributed to germ line mutations in mismatch repair (MMR) genes, somatic mutations of known MMR genes are infrequent in MSI+ sporadic endometrial carcinomas. Recently, cytosine methylation of the MLH1 promoter region has been identified in a subset of MSI+ colon primary carcinomas and cell lines. We studied the MLH1 and MSH2 promoter methylation status in 29 presumably sporadic uterine endometrioid carcinomas (UECs), which had previously been characterized for the MSI phenotype and a subset for DNA MMR gene mutational status. We found that 13 (45%) of 29 cases of EC were hypermethylated in the 5′ CpG island of MLH1. Hypermethylation of MSH2 was not observed. MLH1 was hypermethylated in 12 (92%) of 13 MSI+ tumors, while only 1 (6%) of 16 MSI- tumors (Fischers exact test P<0.0001). Other tumor types we tested did not demonstrate MLH1 promoter hypermethylation. Our data suggest that hypermethylation of MLH1, but not of MSH2, is associated with the MSI phenotype in sporadic endometrial carcinomas.

hMLH1 Promoter Hypermethylation Is an Early Event in Human Endometrial Tumorigenesis

It has recently been suggested that silencing of the hMLH1 gene by promoter hypermethylation is the mechanism underlying the presence of the microsatellite instability (MSI) phenotype in sporadic colon and endometrial carcinomas. To determine whether hMLH1 promoter hypermethylation is a relatively early event in endometrial tumorigenesis we evaluated endometrial hyperplasia (EH) characterized as simple, complex, and atypical (the direct precursor of endometrial carcinoma) for hMLH1 aberrant methylation. In addition, we studied the hMLH1, hMSH2, hMSH3, and hMSH6 promoter methylation and MSI status of those endometrial carcinomas with synchronous hyperplasias and those without them. We found that 11 of 12 (91%) cases of endometrial carcinoma (EC) displaying MSI had hMLH1 promoter hypermethylation, whereas aberrant methylation of any of the other mismatch repair genes was not observed. All 15 cases of EC without MSI were unmethylated at hMLH1. Abnormal methylation of hMLH1 was also present in 8 of 116 (7%) cases of EH and was restricted primarily to the atypical endometrial hyperplasia (AEH) type with coexisting endometrial carcinoma. In this set, half of EH methylated at hMLH1 displayed MSI, whereas none of the unmethylated EH had MSI. Our data suggest that hypermethylation of hMLH1 can be an early event in the pathogenesis of EC, preceding the development of an apparent MSI phenotype in a subset of cases.

Epigenetic inactivation of LKB1 in primary tumors associated with the Peutz-Jeghers syndrome

Germ-line mutations of the LKB1 gene cause Peutz-Jeghers syndrome (PJS) characterized by mucocutaneous pigmentation, predisposition to benign hamartomas of the gastrointestinal tract and also to several types of tumors. However, somatic mutations of this gene are very rare. To examine inactivation of LKB1 by epigenetic mechanisms, we investigated a series of primary tumors and cancer cell lines, for hypermethylation affecting the CpG island located in the 5′ region of the LKB1 gene using Methylation-specific PCR (MSP). First, we screened 51 cancer cell lines. Only three colorectal and one cervical carcinoma cell lines were methylated at LKB1, and loss of the LKB1 transcript was demonstrated. Treatment with the demethylating agent 5-aza-2′-deoxycytidine restored LKB1 expression. To address the incidence of LKB1 epigenetic inactivation in primary tumors, we analysed colorectal, breast, gastric, pancreatic, thyroid, bladder and testicular carcinomas (n=195). Normal tissues from the mentioned organs were unmethylated in this region. Among the described tumors, only one colorectal carcinoma and three testicular tumors displayed LKB1 promoter hypermethylation. Further study of those histological types more commonly associated with PJS, demonstrated that LKB1 promoter hypermethylation was present in five of 11 (45%) papillary breast carcinomas. Finally, in three patients with a strong family story suggestive of PJS disease, abnormal LKB1 methylation was found in four of 22 (18%) hamartomatous polyps lesions. Our findings provide an alternative pathway for inactivation of the LKB1 tumor suppressor gene involving promoter hypermethylation.

Point mutation and homozygous deletion of PTEN/MMAC1 in primary bladder cancers

A new tumor suppressor gene PTEN/MMAC1 was recently isolated at chromosome 10q23 and found to be inactivated by point mutation or homozygous deletion in glioma, prostate and breast cancer. PTEN/MMAC1 was also identified as the gene predisposing to Cowden disease, an autosomal dominant cancer predisposition syndrome associated with an increased risk of breast, skin and thyroid tumors and occasional cases of other cancers including bladder and renal cell carcinoma. We screened 345 urinary tract cancers by microsatellite analysis and found chromosome 10q to be deleted in 65 of 285 (23%) bladder and 15 of 60 (25%) renal cell cancers. We then screened the entire PTEN/MMAC1 coding region for mutation in 25 bladder and 15 renal cell primary tumors with deletion of chromosome 10q. Two somatic point mutations, a frameshift and a splicing variant, were found in the panel of bladder tumors while no mutation was observed in the renal cell carcinomas. To screen for homozygous deletion, we isolated two polymorphic microsatellite repeats from genomic BAC clones containing the PTEN/MMAC1 gene. Using these new informative markers, we identified apparent retention at the gene locus indicative of homozygous deletion of PTEN/MMAC1 in four of 65 bladder and 0 of 15 renal cell tumors with LOH through chromosome 10q. Identification of the second inactivation event in six bladder tumors with LOH of 10q implies that the PTEN/MMAC1 gene is occasionally involved in bladder tumorigenesis. However, the low frequency of biallelic inactivation suggests that either PTEN/MMAC1 is inactivated by other mechanisms or it is not the only target of chromosome 10q deletion in primary bladder and renal cell cancer.

Hypermethylation of the hMLH1 gene promoter is associated with microsatellite instability in early human gastric neoplasia

A significant portion of gastric cancers exhibit defective DNA mismatch repair, manifested as microsatellite instability (MSI). High-frequency MSI (MSI-H) is associated with hypermethylation of the human mut-L homologue 1 (hMLH1) mismatch repair gene promoter and diminished hMLH1 expression in advanced gastric cancers. However, the relationship between MSI and hMLH1 hypermethylation has not been studied in early gastric neoplasms. We therefore investigated hMLH1 hypermethylation, hMLH1 expression and MSI in a group of early gastric cancers and gastric adenomas. Sixty-four early gastric neoplasms were evaluated, comprising 28 adenomas, 18 mucosal carcinomas, and 18 carcinomas with superficial submucosal invasion but clear margins. MSI was evaluated using multiplex fluorescent PCR to amplify loci D2S123, D5S346, D17S250, BAT 25 and BAT 26. Methylation-specific PCR was performed to determine the methylation status of hMLH1. In two hypermethylated MSI-H cancers, hMLH1 protein expression was also evaluated by immunohistochemistry. Six of sixty-four early gastric lesions were MSI-H, comprising 1 adenoma, 4 mucosal carcinomas, and 1 carcinoma with superficial submucosal invasion. Two lesions (one adenoma and one mucosal carcinoma) demonstrated low-frequency MSI (MSI-L). The remaining 56 neoplasms were MSI-stable (MSI-S). Six of six MSI-H, one of two MSI-L, and none of thirty MSI-S lesions showed hMLH1 hypermethylation (P<0.001). Diminished hMLH1 protein expression was demonstrated by immunohistochemistry in two of two MSI-H hypermethylated lesions. hMLH1 promoter hypermethylation is significantly associated with MSI and diminished hMLH1 expression in early gastric neoplasms. MSI and hypermethylation-associated inactivation of hMLH1 are more prevalent in early gastric cancers than in gastric adenomas. Thus, hypermethylation-associated inactivation of the hMLH1 gene can occur early in gastric carcinogenesis.

Inactivation of the tissue inhibitor of metalloproteinases-2 gene by promoter hypermethylation in lymphoid malignancies

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-Hodgkins 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.