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James G Herman

Publications and source records attributed to James G Herman.

At least 37 records · Page 2Linked to original sources
PubMed2005

Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation.

Double-stranded RNA molecules targeted to gene promoter regions can induce transcriptional gene silencing in a DNA cytosine methylation-dependent manner in plants (RNA-dependent DNA methylation). Whether a similar mechanism exists in mammalian systems is a vital and controversial issue. DNA methylation is an important component in mammalian gene silencing for normal processes such as gene imprinting and X-chromosome inactivation, and aberrant CpG island hypermethylation at tumor-suppressor promoters is associated with transcriptional silencing and loss of gene function in cancer. Hence, we investigated whether RNA-dependent DNA methylation might operate in human cancers to mediate epigenetic silencing using the endogenous gene CDH1 as a potential target. The loss of this cell-cell adhesion factor facilitates the metastatic process, and its promoter is frequently hypermethylated in breast and other cancers. We found that, although small double-stranded RNAs targeted exclusively to the CDH1 promoter could effectively induce transcriptional repression with chromatin changes characteristic of inactive promoters, this was entirely independent of DNA methylation. Moreover, we could accomplish such silencing in a cancer cell line genetically modified to lack virtually any capacity to methylate DNA.

Cell Line, Tumor↗
PubMed2005

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-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.

Cell Line, Tumor↗
PubMed2005

CHFR promoter hypermethylation in colon cancer correlates with the microsatellite instability phenotype.

A subset of sporadic colon cancers has been shown to have microsatellite instability caused by an epigenetic inactivation of the MLH1 gene by hypermethylation of the the CpG island in its promoter region. We report here that in colorectal cancer, inactivation of the MLH1 gene is frequently accompanied by hypermethylation of the CpG island in the promoter of the mitotic gene checkpoint with forkhead and ring finger domains (CHFR). This was first observed in the colon cancer cell lines HCT-116, DLD-1, RKO and HT29. Among the 61 primary colon cancer samples studied, hypermethylation of the MLH1 and the CHFR promoter was found in 31% of the tumors. In 68% of all primary cancers (13/19) with MLH1 promoter hypermethylation, hypermethylation of the CHFR promoter was observed as well (P-value < 0.0001, Fisher's two-sided exact). Hypermethylation of the HLTF, MGMT, RASSF1, APC, p14 and p16 promoter regions were also frequent events, being observed in 48% (28/58), 40% (26/64), 21% (14/64), 50% (31/62), 43% (26/60) and 56% (35/63), respectively. However, methylation of these genes was not associated with methylation of either MLH1 or CHFR. The observed methylation profile was unrelated to Duke's stage. The coordinated loss of both mismatch repair caused by methylation of MLH1 and loss of checkpoint control associated with methylation of CHFR suggests the potential to overcome cell cycle checkpoints, which may lead to an accumulation of mutations.

Adaptor Proteins, Signal Transducing↗
PubMed2005

The fundamental role of epigenetics in hematopoietic malignancies.

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.

CpG Islands↗
PubMed2005

Clinical implications of aberrant DNA methylation patterns in acute myelogenous leukemia.

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.

Adult↗
PubMed2005

Can we improve the cytologic examination of malignant pleural effusions using molecular analysis?

BACKGROUND: Currently, 40% of patients remain undiagnosed after routine cytologic examination for malignant pleural effusions. Deoxyribonucleic acid (DNA) methylation is a robust strategy for detecting cancer early in tissue. We hypothesized that DNA methylation would be more sensitive in diagnosing patients with malignant pleural effusions than cytology. METHODS: We conducted a prospective cohort study of 31 inpatients with pleural effusions (24 malignant pleural effusions metastatic from 10 different organs and 7 benign) over 18 months. Aspirated pleural fluid underwent cytologic examination and DNA extraction for nested methylation-specific polymerase chain reaction (PCR). We assayed for promoter hypermethylation in 8 genes known to be methylated in many cancers. Pleural fluid was considered positive if 2 or more genes were methylated by methylation-specific PCR. RESULTS: Cytology alone confirmed malignant pleural effusions in 15 of 24 patients (sensitivity 63%), whereas methylation alone positively identified 16 of 24 patients (sensitivity 67%). Both tests had 100% specificity in predicting benign effusions. If cytology and methylation were considered together, they exhibited 88% sensitivity and 100% specificity in discriminating benign and malignant effusions. Combined, the two assays were more sensitive than either test alone. Although the positive predictive value of each test was 100%, the negative predictive value of cytology and methylation combined was 78%, better than 47% and 44% for methylation and cytology alone, respectively. CONCLUSIONS: Epigenetic analysis of pleural fluid can detect malignant DNA from a variety of neoplasms, provide complementarity with cytology, and improve the diagnostic yield of the current standard examination of pleural fluid.

Breast Neoplasms↗
PubMed2005

Surgical resection of limited disease small cell lung cancer in the new era of platinum chemotherapy: Its time has come.

OBJECTIVE: Although resection is not the standard of care in treating small cell lung cancer, new platinum drugs and modern staging have allowed the role of surgery to be reevaluated. METHODS: We reviewed our institutional experience of 1415 patients with small cell lung cancer from 1976 to 2002 among whom 82 (6%) underwent surgery with curative intent. RESULTS: Median age at surgery was 62 years, and small cell lung cancer of mixed morphology represented 14 of 82 (17%). Treatment consisted of surgery alone in 11% of cases (9/82), surgery with neoadjuvant therapy in 22% (18/82), and surgery with adjuvant therapy in 55% (45/82). Prophylactic cranial irradiation was given to 23% (19/82). The 5-year survival of the entire cohort was 42%. The 5-year survival of patients receiving adjuvant chemotherapy (n = 41) was significantly different according to whether patients had received platinum or nonplatinum regimens (68% vs 32.2%, P = .04). Among patients with stage I disease who received adjuvant chemotherapy (n = 24), the 5-year survivals for patients receiving platinum and nonplatinum chemotherapy were 86% and 42%, respectively ( P < .02). If patients who received either neoadjuvant or adjuvant therapy (n = 56) were considered, the 5-year survival was significantly better for platinum than for nonplatinum chemotherapy (62% vs 36%, P = .05). The 5-year survival was also better for those undergoing lobectomies (n = 52) than for those with limited resections (n = 15, 50% vs 20%, P = .03). Survival outcomes also differed by gender, with female patients having a 5-year survival advantage over male patients (60% vs 28%, P = .004). CONCLUSION: These results support a reevaluation of the role of surgery in the multimodality therapy for small cell lung cancer, which currently includes only radiotherapy and chemotherapy.

Adult↗
PubMed2005

Promoter methylation profiles of tumor suppressor genes in intrahepatic and extrahepatic cholangiocarcinoma.

Recent studies indicate that tumor suppressor genes can be epigenetically silenced through promoter hypermethylation. To further understand epigenetic alterations in cholangiocarcinoma, we have studied the methylation profiles of 12 candidate tumor suppressor genes (APC, E-cadherin/CDH1, MGMT, RASSF1A, GSTP, RAR-beta, p14ARF, p15INK4b, p16INK4a, p73, hMLH1 and DAPK) in 72 cases of cholangiocarcinoma, including equal number cases of intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma. A total of 10 cases of benign biliary epithelia were included as controls. The methylation status of tumor suppressor genes was analyzed using methylation-specific PCR. We found that 85% of all cholangiocarcinomas had methylation of at least one tumor suppressor gene. The frequency of tumor suppressor gene methylation in cholangiocarcinoma was: RASSF1A (65%), p15INK4b (50%), p16INK4a (50%), APC (46%), E-cadherin/CDH1 (43%), p14(ARF) (38%), p73 (36%), MGMT (33%), hMHL1 (25%), GSTP (14%), RAR-beta (14%) and DAPK (3%). Although single tumor suppressor gene methylation can be seen in benign biliary epithelium, methylation of multiple tumor suppressor genes is only seen in cholangiocarcinoma. About 70% (50/72) of the cholangiocarcinomas had three or more tumor suppressor genes methylated and 52% (38/72) of cases had four or more tumor suppressor genes methylated. Concerted methylation of multiple tumor suppressor genes was closely associated with methylation of RASSF1A, p16 and/or hMHL1. Methylation of RASSF1A was more common in extrahepatic cholangiocarcinoma than intrahepatic cholangiocarcinoma (83 vs 47%, P=0.003) while GSTP was more frequently seen in intrahepatic compared to extrahepatic cholangiocarcinoma (31 vs 6%, P=0.012). Our study indicates that methylation of promoter CpG islands of tumor suppressor genes is a common epigenetic event in cholangiocarcinoma. Based on distinct methylation profiles, intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma are two closely related but biologically unique neoplastic processes. Taking advantage of the unique concurrent methylation profile of multiple genes in cholangiocarcinoma may facilitate the distinction of cholangiocarcinoma from benign biliary epithelium in clinical settings.

Bile Duct Neoplasms↗
PubMed2005

Methylation-specific polymerase chain reaction.

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.

Base Sequence↗
PubMed2004

Epigenetic regulation of protein phosphatase 2A (PP2A), lymphotactin (XCL1) and estrogen receptor alpha (ER) expression in human breast cancer cells.

Absence of the estrogen receptor alpha (ER) in human breast cancer cells is an indicator of poor prognosis, and predictive of lack of response to hormonal therapy. Previous studies in our laboratory and others have shown that epigenetic regulation, including DNA methylation and histone deacetylation, are common mechanisms leading to ER gene silencing. Through the use of pharmacologic inhibitors, 5-aza 2'deoxycytidine (AZA) and Trichostatin A (TSA), we have shown that alterations in both of these mechanisms results in synergistic reexpression of ER mRNA and functional protein. These alterations may play a larger role in stimulation of cell signaling pathways leading to ER expression. We have utilized newly developed genome wide screening microarray techniques to identify gene(s) contributing to the hormone independent phenotype and AZA/TSA mediated ER expression. From this screen, we identified and confirmed expression of 4 candidate genes (PP2A, XCL1, THY1 and NBC4) as potential regulators of the hormone independent phenotype. Expression of two genes, XCL1 and PP2A, appeared to be correlated with ER expression. PP2A expression was not changed with ER degradation using ICI 182,780 whereas XCL1 expression decreased in the presence of AZA/TSA and ICI 182,780. This suggests that PP2A may be a determinant of ER expression while XCL1 appears to be ER responsive and downstream of ER expression. These gene products may be novel targets to be further explored in the development of new therapeutics for ER negative breast cancer.

Acetylation↗
PubMed2004

Hypermethylation of the GATA genes in lung cancer.

PURPOSE: In lung cancer, DNA hypermethylation is known to be a common event. EXPERIMENTAL DESIGN: Gene expression and methylation status of GATA-4, GATA-5, and GATA-6 were analyzed with cell lines and primary human lung cancers. Methylation profiles of primary lung cancers were analyzed and correlated with clinical as well as histopathological data. RESULTS: Complete methylation was detected by methylation-specific PCR for both GATA-4 and GATA-5 in four cell lines (H358, DMS-53, A549, and H1299), all of which had no expression by reverse transcription-PCR analysis. Demethylation with 5-aza-2'deoxycytidine restored expression in each case. GATA-6 was ubiquitously expressed in all of the six cell lines. GATA-4 bisulfite sequencing revealed complete methylation of the GATA-4 promoter in H358 cells, correlating well with its lack of expression at the mRNA level. Only a few CpG sites showed methylation by bisulfite sequencing within the GATA-4 promoter in a cell line that expressed the gene. In 63 cases of primary lung cancers, GATA-4 and GATA-5 promoter methylation was detected in (42 of 63) 67% and (26 of 63) 41%, respectively. GATA-6 remained unmethylated both in cell lines and primary tumors. Six autopsy specimens of normal lung tissue showed no aberrant promoter hypermethylation for the GATA genes. Correlation of concomitant GATA-4 and GATA-5 methylation with clinicopathological parameters only found a statistically significant increase in methylation frequency with increasing patient age (P < 0.001). CONCLUSIONS: These epigenetic changes in the GATA genes in lung cancer are tumor-specific, relate to the loss of GATA gene expression, and occur increasingly in the elderly.

Adenocarcinoma↗
PubMed2004

Tumor cell-specific BRCA1 and RASSF1A hypermethylation in serum, plasma, and peritoneal fluid from ovarian cancer patients.

Because existing surgical and management methods can consistently cure only early-stage ovarian cancer, novel strategies for early detection are required. Silencing of tumor suppressor genes such as p16INK4a, VHL, and hMLH1 have established promoter hypermethylation as a common mechanism for tumor suppressor inactivation in human cancer and as a promising target for molecular detection in bodily fluids. Using sensitive methylation-specific PCR, we screened matched tumor, preoperative serum or plasma, and peritoneal fluid (washes or ascites) DNA obtained from 50 patients with ovarian or primary peritoneal tumors for hypermethylation status of the normally unmethylated BRCA1 and RAS association domain family protein 1A tumor suppressor genes. Hypermethylation of one or both genes was found in 34 tumor DNA (68%). Additional examination of one or more of the adenomatous polyposis coli, p14ARF, p16INK4a, or death associated protein-kinase tumor suppressor genes revealed hypermethylation in each of the remaining 16 tumor DNA, which extended diagnostic coverage to 100%. Hypermethylation was observed in all histologic cell types, grades, and stages of ovarian tumor examined. An identical pattern of gene hypermethylation was found in the matched serum DNA from 41 of 50 patients (82% sensitivity), including 13 of 17 cases of stage I disease. Hypermethylation was detected in 28 of 30 peritoneal fluid DNA from stage IC-IV patients, including 3 cases with negative or atypical cytology. In contrast, no hypermethylation was observed in nonneoplastic tissue, peritoneal fluid, or serum from 40 control women (100% specificity). We conclude that promoter hypermethylation is a common and relatively early event in ovarian tumorigenesis that can be detected in the serum DNA from patients with ovary-confined (stage IA or B) tumors and in cytologically negative peritoneal fluid. Analysis of tumor-specific hypermethylation in serum DNA may enhance early detection of ovarian cancer.

Adolescent↗
PubMed2004

Hypermethylation in histologically distinct classes of breast cancer.

PURPOSE: A number of different genes are known to be inactivated by aberrant hypermethylation in breast cancer, but it is still unknown to what extent these epigenetic alterations differ according to specific breast cancer phenotypes. We sought to determine whether the extent of hypermethylation or defined profiles of gene hypermethylation are associated with biological characteristics of breast cancers. EXPERIMENTAL DESIGN: We evaluated methylation status of 12 different genes in a series of 109 invasive breast tumors, representing the ductal, lobular, and mucinous histologic subtypes using methylation-specific PCR. Frequencies of methylation were compared across the recognized histologic classes, and multivariate techniques (latent class analysis, factor analysis, recursive partitioning, and hierarchical clustering) were used to seek patterns of methylation for individual genes that distinguish recognized histologic types of breast cancer or define breast cancer phenotypes on a molecular level. RESULTS: All 109 cases studied have aberrant methylation of multiple genes (3 to 10 genes per case), demonstrating that gene hypermethylation is pervasive in breast cancer. Lobular cancers and mucinous cancers, which often have relatively low levels of chromosomal changes, have higher overall frequencies of hypermethylation than ductal cancers (49% in lobular and mucinous versus 40% in ductal), but there is a relatively unimodal distribution of methylation frequency for all three histologic types. Only one of the individual genes studied, BRCA1, has a variable frequency of methylation that is significantly dependent on histologic pattern of tumor growth, with a higher frequency of methylation in mucinous cancers than ductal or lobular cancers. Although some trends of histology-specific gene methylation were seen, methylation patterns could not definitively classify breast cancers according to histologic type. CONCLUSIONS: Although a more comprehensive hypermethylation profile could potentially be useful for breast cancer classification and understanding the biology of this disease, it appears that the hypermethylation patterns across various forms of breast cancer are less distinct than those between breast cancer and cancers of different tissue origins. Furthermore, the relatively unimodal distribution of methylation frequency for all three histologic types does not support there being a distinct CpG island methylator phenotype for breast cancer.

Adult↗
PubMed2004

Promoter hypermethylation of resected bronchial margins: a field defect of changes?

PURPOSE: Histologically positive bronchial margins after resection for non-small cell lung cancer are associated with shortened patient survival due to local recurrence. We hypothesized that DNA promoter hypermethylation changes at bronchial margins could be detected in patients with no histological evidence of malignancy and that they would reflect epigenetic events in the primary tumor. EXPERIMENTAL DESIGN: Bronchial margins, primary tumor, bronchoalveolar fluid, and paired nonmalignant lung were obtained from 20 non-small cell lung cancer patients who underwent a lobectomy or greater resection. Disease-specific recurrence was the primary end point. The methylation status of p16, MGMT, DAPK, SOCS1, RASSF1A, COX2, and RARbeta was examined using methylation-specific polymerase chain reaction. RESULTS: All malignancies had methylation in at least one locus. Concordance of one gene with an identical epigenetic change in the tumor or bronchial margin was observed in 85% of patients. Only one patient had methylation at the bronchial margin for a gene that was not methylated in the corresponding tumor. Median time to recurrence was 37 months (range, 5-71 months). There were two local recurrences and five metastases. There were no significant correlations between DNA methylation in tumor, margins, or bronchoalveolar fluid specimens and either regional recurrence or distant metastases. CONCLUSIONS: Histologically negative bronchial margins of resected non-small cell lung cancer exhibit frequent hypermethylation changes in multiple genes. These hyper-methylation abnormalities are also present in the primary tumor and thus may represent a field defect of preneoplastic changes that occurs early in carcinogenesis.

Adult↗
PubMed2004

The clinical application of targeting cancer through histone acetylation and hypomethylation.

Methods of gene inactivation include genetic events such as mutations or deletions. Epigenetic changes, heritable traits that are mediated by changes in DNA other than nucleotide sequences, play an important role in gene expression. Two epigenetic events that have been associated with transcriptional silencing include methylation of CpG islands located in gene promoter regions of cancer cells and changes in chromatin conformation involving histone acetylation. Recent evidence demonstrates that these processes form layers of epigenetic silencing. Reversal of these epigenetic processes and up-regulation of genes important to prevent or reverse the malignant phenotype has therefore become a new therapeutic target in cancer treatment.

Acetylation↗
PubMed2004

Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer.

Aberrant WNT pathway signaling is an early progression event in 90% of colorectal cancers. It occurs through mutations mainly of APC and less often of CTNNB1 (encoding beta-catenin) or AXIN2 (encoding axin-2, also known as conductin). These mutations allow ligand-independent WNT signaling that culminates in abnormal accumulation of free beta-catenin in the nucleus. We previously identified frequent promoter hypermethylation and gene silencing of the genes encoding secreted frizzled-related proteins (SFRPs) in colorectal cancer. SFRPs possess a domain similar to one in the WNT-receptor frizzled proteins and can inhibit WNT receptor binding to downregulate pathway signaling during development. Here we show that restoration of SFRP function in colorectal cancer cells attenuates WNT signaling even in the presence of downstream mutations. We also show that the epigenetic loss of SFRP function occurs early in colorectal cancer progression and may thus provide constitutive WNT signaling that is required to complement downstream mutations in the evolution of colorectal cancer.

Cell Line, Tumor↗
PubMed2004

Hypermethylation of a small CpGuanine-rich region correlates with loss of activator protein-2alpha expression during progression of breast cancer.

The transcription factor activator protein-2alpha (AP-2alpha) has recently been implicated as a tumor suppressor protein that can be lost during tumor progression and that exhibits growth-inhibitory properties when overexpressed in cancer cell lines. We now demonstrate that hypermethylation of a discrete 5' region within a promoter CpG island of the gene is associated in breast cancer with the loss of AP-2alpha expression. Multiple CpG sites within the island become hypermethylated during breast cancer evolution. However, only hypermethylation of the most CpG-rich region, a small, approximately 300-bp area at the 3' end of exon 1, fully distinguishes neoplastic from normal breast tissue and correlates with transcriptional silencing. In cell culture, silenced AP-2alpha, associated with exon 1 hypermethylation, is re-expressed by 5-aza-2'deoxycytidine resulting in the restoration of a functional DNA sequence-specific binding protein. In vivo, as detected by a very sensitive nested PCR approach, methylation of the discrete AP-2alpha exon 1 region does not occur in normal breast epithelium and occurs in only 3 (16%) of 19 ductal carcinoma in situ (DCIS) lesions, but is present in 12 (75%) of 16 invasive breast tumors (P < 0.001; DCIS versus invasive cancers). Tumors unmethylated for this region expressed AP-2alpha protein throughout, whereas tumors with hypermethylation showed large areas of loss. Our studies then determine that hypermethylation of a small region of a CpG island correlates with silencing of AP-2alpha in breast cancer and suggest that inactivation of this gene could be a factor in, and a useful marker for, the progression of DCIS lesions.

Breast Neoplasms↗