Geneviève Clément

Methylation of APC, TIMP3, and TERT: a new predictive marker to distinguish Barrett's oesophagus patients at risk for malignant transformation.

Barretts associated oesophageal adenocarcinoma (EAC) is one of the most rapidly increasing malignancies in Western countries. Because of its poor prognosis, management of this disease through screening of Barretts oesophagus (BE) patients and identification of those with a high risk of developing an adenocarcinoma seems a promising approach. Early molecular markers of malignant transformation might contribute to such screening approaches. Gene promoter methylation analysis was performed on normal, pre‐neoplastic, and neoplastic lesions from BE patients. All lesions of interest were sampled by microdissection from formalin‐fixed paraffin‐embedded tissue sections. We found that, in 27 adenocarcinomas, APC, TIMP3, TERT, CDKN2A, and SFRP1 promoters were methylated in 93%, 65%, 64%, 48%, and 91%, respectively; in contrast MLH1, RASSF1, RARB, CDH1, and FHIT promoters were methylated in less than 5% of the tumours. In BE mucosa from patients who had progressed to adenocarcinoma (12 samples), APC, TIMP3, and TERT promoters were hypermethylated in 100%, 91%, and 92% of cases, whereas in BE mucosa from patients who had not progressed (16 samples) methylation was found only in 36%, 23%, and 17%, respectively. Furthermore, the epigenetic profile of BE with and without EAC differed significantly with, respectively, 81% and 26% of the PCR samples showing promoter hypermethylation for APC, TIMP3, and TERT (p < 0.0001). Promoter methylation of CDKN2A was infrequently detected in BE samples, while SFRP1 methylation was observed in all samples. Our results suggest that promoter methylation profiling of BE using multiple target genes including APC, TIMP3, and TERT might be used as a predictive marker for increased EAC risk. Copyright

Alterations of the Wnt signaling pathway during the neoplastic progression of Barrett's esophagus

Aberrant activation of the Wnt signaling pathway has been reported during neoplastic progression in Barretts esophagus (BE). However, mutations in APC and CTNNB1 genes were rarely observed. In this study, expression pattern of Wnt ligands, Frizzled receptors and APC, as well as the methylation status of the APC, SFRP1 and SFRP2 promoter genes were investigated in normal esophageal mucosa and in preneoplastic and neoplastic lesions of BE patients. Promoter methylation of APC was found in all BE samples and in 95% of esophageal adenocarcinomas (EAC). Full methylation of APC correlated with lack of expression. In EAC, nuclear translocation of β-catenin was observed regardless of the expression of APC. WNT2 expression was higher in dysplasia and EAC than in BE, with 20/26 (77%) of the EAC showing high expression of WNT2. SFRP1 methylation occurred in all BE samples and in 96% of EAC, while SFRP2 was methylated in 73% of the normal squamous esophageal mucosa samples. In conclusion, (1) alterations of key regulators of the Wnt signaling are frequent in the pathogenesis of BE; (2) the APC and SFRP1 genes are inactivated by promoter methylation in BE; (3) the WNT2 gene is upregulated along the progression from low-grade dysplasia to EAC.

Epigenetic alteration of the Wnt inhibitory factor-1 promoter occurs early in the carcinogenesis of Barrett's esophagus

The role of Wnt antagonists in the carcinogenesis of esophageal adenocarcinoma (EAC) remains unclear. We hypothesized that downregulation of the Wnt inhibitory factor‐1 (WIF‐1) might be involved in the neoplastic progression of Barretts esophagus (BE). We analyzed the DNA methylation status of the WIF‐1 promoter in normal, preneoplastic, and neoplastic samples from BE patients and in EAC cell lines. We investigated the role of WIF‐1 on EAC cell growth and the chemosensitization of the cells to cisplatin. We found that silencing of WIF‐1 correlated with promoter hypermethylation. EAC tissue samples showed higher levels of WIF‐1 methylation compared to the matched normal epithelium. In addition, we found that WIF‐1 hypermethylation was more frequent in BE samples from patients with EAC than in BE samples from patients who had not progressed to EAC. Restoration of WIF‐1 in cell lines where WIF‐1 was methylation‐silenced resulted in growth suppression. Restoration of WIF‐1 could sensitize the EAC cells to the chemotherapy drug cisplatin. Our results suggest that silencing of WIF‐1 through promoter hypermethylation is an early and common event in the carcinogenesis of BE. Restoring functional WIF‐1 might be used as a new targeted therapy for the treatment of this malignancy. (Cancer Sci 2008; 99: 46–53)

Inhibition of Wnt-2 and galectin-3 synergistically destabilizes β-catenin and induces apoptosis in human colorectal cancer cells

Constitutive activation of the Wnt pathway as a result of APC, AXIN1 or CTNNB1 mutations has been found in most colorectal cancers. For a long time, this aberrant Wnt activation has been thought to be independent of upstream signals. However, recent studies indicate that upstream signals retain their ability to regulate the Wnt pathway even in the presence of downstream mutations. Wnt‐2 is well known for its overexpression in colorectal cancer. Galectin‐3 (Gal‐3), a multifunctional carbohydrate binding protein implicated in a variety of biological functions, has recently been reported to interact with β‐catenin. In this study, we investigated roles of Wnt‐2 and Gal‐3 in the regulation of canonical Wnt/β‐catenin signaling. We found that siRNA silencing of either Wnt‐2 or Gal‐3 expression inhibited TCF‐reporter activity, decreased cytosolic β‐catenin level and induced apoptosis in human colorectal cancer cells containing downstream mutations. More interestingly, we showed that inhibition of both Wnt‐2 and Gal‐3 had synergistic effects on suppressing canonical Wnt signaling and inducing apoptosis, suggesting that aberrant canonical Wnt/β‐catenin signaling in colorectal cancer can be regulated at multiple levels. The combined inhibition of Wnt‐2 and Gal‐3 may be of superior therapeutic advantage to inhibition by either one of them, giving rise to a potential development of novel drugs for the targeted treatment of colorectal cancer.

Inhibition of Hsp90 leads to cell cycle arrest and apoptosis in human malignant pleural mesothelioma.

Introduction: Heat shock protein 90 (Hsp90) is an abundant molecular chaperone that mediates the maturation and stability of a variety of proteins associated with the promotion of cell growth and survival. Inhibition of Hsp90 function leads to proteasomal degradation of its mis-folded client proteins. Recently, Hsp90 has emerged as being of prime importance to the growth and survival of cancer cells and its inhibitors have already been used in phase I and II clinical trials. Methods: We investigated how 17-allylamino-17-demethoxygeldanamycin (17-AAG), a small molecule inhibitor of Hsp90, is implicated in human malignant pleural mesothelioma (MM). Results: We found that 17-AAG led to significant G1 or G2/M cell cycle arrest, inhibition of cell proliferation, and decrease of AKT, AKT1, and survivin expression in all human malignant pleural mesothelioma cell lines examined. We also observed significant apoptosis induction in all MM cell lines treated with 17-AAG. Furthermore, 17-AAG induced apoptosis in freshly cultured primary MM cells and caused signaling changes identical to those in 17-AAG treated MM cell lines. Conclusion: These results suggest that Hsp90 is strongly associated with the growth and survival of MM and that inhibition of Hsp90 may have therapeutic potential in the treatment of MM.

EMX2 is epigenetically silenced and suppresses growth in human lung cancer

Lung cancer is a common cancer and the leading cause of cancer-related death worldwide. Aberrant activation of WNT signaling is implicated in lung carcinogenesis. EMX2, a human homologue of the Drosophila empty spiracles gene is a homeodomain-containing transcription factor. The function of EMX2 has been linked to the WNT signaling pathway during embryonic patterning in mice. However, little is known about the role of EMX2 in human tumorigenesis. In this study, we found that EMX2 was dramatically downregulated in lung cancer tissue samples and this downregulation was associated with methylation of the EMX2 promoter. Restoration of EMX2 expression in lung cancer cells lacking endogenous EMX2 expression suppressed cell proliferation and invasive phenotypes, inhibited canonical WNT signaling, and sensitized lung cancer cells to the treatment of the chemo cytotoxic drug cisplatin. On the other hand, knockdown of EMX2 expression in lung cancer cells expressing endogenous EMX2 promoted cell proliferation, invasive phenotypes and canonical WNT signaling. Taken together, our study suggests that EMX2 may have important roles as a novel suppressor in human lung cancer.

Monoallelic Methylation of the APC Promoter Is Altered in Normal Gastric Mucosa Associated with Neoplastic Lesions

Adenomatous polyposis coli (APC) promoter hypermethylation has been reported frequently in normal gastric mucosa, but it remained to be clarified whether this occurs in every individual. In this study, methylation of the APC promoter was analyzed in histologically normal-appearing gastric mucosa samples by methylation-sensitive single-strand conformation analysis and by a methylation-sensitive dot blot assay. Epithelial cell samples were collected by microdissection from tissue sections. Equal amounts of methylated and unmethylated APC alleles were found in all gastric mucosa samples from patients without any gastric lesions (20 samples). Allele-specific methylation analysis showed that the methylation of the APC promoter was monoallelic; however, which allele was methylated depended on the cell type. Increased or decreased methylation was found in 10 of 36 (28%) normal gastric mucosa samples adjacent to a gastric or esophageal adenocarcinoma. No allelic loss was found at the APC locus. Modification of the methylation status was also found in 3 of 21 (14%) normal-appearing gastric mucosa samples adjacent to intestinal metaplasia. In contrast, all normal mucosa samples in cases with chronic gastritis but without metaplasia or dysplasia showed a monoallelic methylation pattern. Our results indicate the following: (a) In normal gastric mucosa, the APC promoter shows monoallelic methylation, which is not due to imprinting but most likely due to allelic exclusion; (b) the excluded allele differs between foveolar and glandular epithelial cells; (c) the APC methylation pattern is frequently altered in normal-appearing gastric mucosa of gastric or esophageal adenocarcinoma patients; and (d) such alterations also occur in normal gastric mucosa adjacent to intestinal metaplasia.

Methylation of CpG island promoters in uveal melanoma

Background: Inactivation of tumour-related genes by promoter hypermethylation is a common epigenetic event in the development of a variety of tumours. Aim: To investigate in primary uveal melanoma the status of promoter methylation of genes thought to be involved in tumour development: p16, TIMP3, RASSF1, RARB, FHIT, hTERT and APC. Methods: Gene promoter methylation was studied by methylation-sensitive single-strand conformation analysis and dot-blot assay in a series of 23 primary uveal melanomas. All DNA samples were obtained from paraffin-embedded formalin-fixed tissue blocks. Results: hTERT promoter methylation was found with a relatively high frequency (52%). Promoter methylation of p16, TIMP3, RASSF1, RARB, FHIT and APC was a rare event. For none of these genes did promoter methylation exceed 15% of tumour samples, and, for some genes (FHIT and APC), no methylation was found at all. Furthermore, promoter methylation was absent in 39% (9/23) of cases. In only 22% (5/23) of cases was hypermethylation of at least two promoters observed. Conclusions: Promoter methylation of hTERT is a regular event in uveal melanoma. Hypermethylation of the other genes studied does not seem to be an essential element in the development of this tumour. As promoter methylation of APC, RASSF1 and RARB is often observed in cutaneous melanoma, these results suggest that different epigenetic events occur in the development of cutaneous and uveal melanoma.

Targeting the Wnt signaling pathway to treat Barrett’s esophagus

Barrett’s esophagus (BE) is an acquired condition in which the normal squamous epithelium in the distal esophagus is replaced by a metaplastic columnar epithelium, as a complication of chronic gastroesophageal reflux. The clinical significance of this disease is its associated predisposition to esophageal adenocarcinoma (EAC). Recently, and similarly to other human malignancies, the Wnt signaling pathway and its key component β-catenin have been implicated in the carcinogenesis of BE. Although mutations in adenomatous polyposis coli (APC) or β-catenin are rare in EAC, alterations of upstream components, such as overexpression of Wnt2 ligand or downregulation of Wnt antagonists may play dominant roles in the activation of the Wnt pathway. Increasing evidence suggests that inhibiting the Wnt pathway may be a new targeted therapy for the treatment of cancers and could, therefore, be promising for the cure of EAC, which remains a highly lethal disease.

Methylation-Sensitive Single-Strand Conformation Analysis

: The last few years have seen a growing interest in the study of DNA methylation because of its now acknowledged implication in cancer. The use of bisulfite to convert unmethylated cytosine to uracil, even as methylated cytosine remains unchanged, constitutes the basis for differentiating between methylated and unmethylated specific CpG sites in CpG islands. This technique therefore is critical to the success of this approach. Different parameters have to be considered in order to achieve a total conversion of cytosines to uracils. Several bisulfite-based methods are available for analyzing DNA methylation status. Methylation-sensitive single-strand conformation analysis (MS-SSCA) yields specific and semiquantitative data. The method is based on bisulfite treatment of DNA followed by polymerase chain reaction using primers without a CpG site to avoid selective amplification of either methylated or unmethylated DNA, and finally by single-strand conformation analysis (SSCA). The method allows one to establish clonal variations in the DNA methylation status for clones representing as little as 5-10% of the total cell population. MS-SSCA has, furthermore, a broad application field since it is the appropriate method for the analysis of frozen, fixed, and even microdissected tissues.