E-cadherin Promoter Methylation Research Articles

Calreticulin enhances gastric cancer metastasis by dimethylating H3K9 in the E-cadherin promoter region mediating by G9a

The latest study shows that gastric cancer (GC) ranked the fifth most common cancer (5.6%) with over 1 million estimated new cases annually and the fourth most common cause of cancer death (7.7%) globally in 2020. Metastasis is the leading cause of GC treatment failure. Therefore, clarifying the regulatory mechanisms for GC metastatic process is necessary. In the current study, we discovered that calreticulin (CALR) was highly expressed in GC tissues and related to lymph node metastasis and patient’s terrible prognosis. The introduction of CALR dramatically promoted GC cell migration in vitro and in vivo, while the repression of CALR got the opposite effects. Cell migration is a functional consequence of the epithelial-mesenchymal transition (EMT) and is related to adhesion of cells. Additionally, we observed that CALR inhibition or overexpression regulated the expression of EMT markers (E-cadherin, ZO-1, Snail, N-cadherin, and ZEB1) and cellular adhesive moleculars (Fibronectin, integrin β1and MMP2). Mechanistically, our data indicated that CALR could mediate DNA methylation of E-cadherin promoter by interacting with G9a, a major euchromatin methyltransferase responsible for methylation of histone H3 on lysine 9(H3K9me2) and recruiting G9a to the E-cadherin promoter. Knockdown of G9a in CALR overexpressing models restored E-cadherin expression and blocked the stimulatory effects of CALR on GC cell migration. Taken together, these findings not only reveal critical roles of CALR medicated GC metastasis but also provide novel treatment strategies for GC.

Abstract 2310: Identification of novel essential genes for prostate cancer metastasis by genome scale CRISPR approaches

Abstract Background: Thanks to the availability of new biomarkers, Prostate cancer (PCa) can now be diagnosed earlier and, as a result, PCa patients have a high 5-year survival rate. Nonetheless, the situation changes completely for patients who develop metastasis, for whom 5-year survival rate decreases to 29%. Recent candidate-gene approaches have identified new candidates, but there are still no drugs to prevent or cure metastatic PCa. Hence, there is an urgent need to find effective therapeutic targets. Methodology: In this study we have conducted a high-throughput CRISPR/Cas9 screening in highly metastatic PCa cell lines PC3 and DU145, using the CRISPR/Cas9 knock-out (GeCKO) library, that targets 18,080 genes with 64,751 unique gRNAs. To assess whether each gene loss-of-function may be critical for metastasis development, invasion assays in Matrigel-coated Boyden Chamber membranes were used. Next-generation sequencing and bioinformatic MaGECK analysis were then carried out to identify novel key genes in the development of PCa metastasis. siRNA approach was used to further validate the best candidates. Results: We found 29 candidates and several signaling pathways capable of significatively impair the invasion of, both, PC3 and DU145 cell lines when knocked-out, being PRMT7 the strongest regulator. PRMT7 gene encodes a type II methyltransferase involved in several cellular processes such as mRNA splicing or DNA repair. There are evidences that indicate it regulates EMT through methylation of E-cadherin promoter and other targets. Moreover, its overexpression correlates with higher levels of expression of MMP9 and MMP2 that lead to metastasis appearance in other types of tumors. Our results inhibiting PRMT7 expression with siRNA validated our high-throughput CRISPR/Cas9 screening results and confirmed the implication of PRMT7 in PCa invasion. Conclusions: We have conducted a novel high-throughput CRISPR-Cas9 screening, finding promising gene candidates, such as PRMT7, which, in the future could be used as therapeutical targets to prevent metastasis development in PCa patients. Furthermore, in this study we have also found genes that had been previously associated to PCa metastasis in other candidate-gene studies, giving a proof-of-concept for the use of high performance of CRISPR-Cas9 screenings for the search of candidate genes involved in PCa metastasis. Citation Format: Maria Rodrigo Faus, Sara Manzano Figueroa, Jingchun Qu, Huiqi Qu, Renata Pellegrino, Hakon Hakonarson, Paloma Bragado, Almudena Porras, Alvaro Gutierrez-Uzquiza. Identification of novel essential genes for prostate cancer metastasis by genome scale CRISPR approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2310.

Methylation of Ecadherin gene is correlated with increased risk of nasopharyngeal carcinoma: A meta-analysis

Background: The objective of this study was to estimate the correlation between the Ecadherin (CDH1) promoter methylation and the risk of nasopharyngeal cancer.
 Methods: Based on previous online articles for the evaluation the hypermethylated status of CDH1 gene at the promoter region with nasopharyngeal carcinoma, two independent reviewers selected studies through databases on PubMed, Google Scholar from 2001 to 2014. The software MedCalc® version 18.11 was applied for calculating pooled odd ratios (OR) with levels of data heterogeneity by the fixed and random effects models.
 Results: Of a total of 99 articles, 12 studies with 508 clinical samples of nasopharyngeal carcinoma patients and 282 normal samples were selected in the systematic review for meta-analysis. Overall, the results demonstrated the highly significant association between CDH1 promoter methylation with nasopharyngeal carcinoma under the fixed effects model (OR = 16.155, 95% CI: 8.533 - 30.585, p

Association of low-dose exposure to persistent organic pollutants with E-cadherin promoter methylation in healthy Koreans

Background: Persistent organic pollutants (POPs), despite their considerably low levels in humans, are an increasing concern for the general populations given their various adverse health problems, including metabolic and carcinogenic effects. DNA methylation deregulation is thought to be a key mechanism in the development of human chronic diseases including cancer.Methods: In an attempt to identify biomarkers monitoring low-dose exposure and hazard, we explored whether organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) may influence the methylation of tumour suppressor gene E-cadherin (CDH1) using peripheral blood cells from 364 healthy Korean subjects.Results: CDH1 methylation was observed in 78.3% of study subjects. Serum concentrations of OCPs or PCBs compounds were higher in CDH1 methylation-positive subjects than in methylation-negative ones. After adjusting for various covariates, the odds ratio of CDH1 methylation of the summary measure of PCBs were 1.0, 2.5 (95% confidence interval: 1.2–5.3), 3.6 (1.6–8.1), 3.6 (1.4–8.6), and 2.5 (1.1–5.7) across quintiles of PCBs (Ptrend = 0.01). The values of OCPs were 1.0, 0.9, 1.2, 2.4 (1.0–5.9), and 1.7 (Ptrend = 0.05).Conclusions: In this exploratory study with a small sample, CDH1 methylation might be served as the epigenetic biomarker associated with POPs exposure and adverse health effect.

Chlamydia trachomatis Infection Is Associated with E-Cadherin Promoter Methylation, Downregulation of E-Cadherin Expression, and Increased Expression of Fibronectin and α-SMA-Implications for Epithelial-Mesenchymal Transition.

Chlamydia trachomatis (Ct) can induce scarring disease of the ocular mucosa, known as trachoma, the most common infectious cause of blindness worldwide. We hypothesized that epithelial-mesenchymal transition (EMT) contributes to the fibrotic process in trachomatous scarring. Infection of human conjunctival epithelial cells (HCjE) with Ct activated signaling pathways involved in EMT induction, which was correlated with decreased expression of E-cadherin, guardian of the epithelial phenotype. In addition, Ct infection was associated with increased expression of two mesenchymal cell markers: fibronectin and α-SMA. The DNA methylation statuses of selected regions of E-cadherin, fibronectin, and α-SMA genes revealed that Ct infection was accompanied with changes in DNA methylation of the E-cadherin promoter, while the expression of the two mesenchymal markers was not related with this epigenetic event. Our data suggest that Ct infection of conjunctival epithelial cells induces EMT-like changes that go along with modification of the methylation profile of the E-cadherin promoter and could, as one of the earliest events, contribute to processes triggering conjunctival scarring.

Aberrant gene promoter methylation of E-cadherin, p16 INK4a , p14 ARF , and MGMT in Epstein–Barr virus-associated oral squamous cell carcinomas

The etiology of oral carcinogenesis appears to be multifactorial. There is emerging evidence of the presence of Epstein-Barr virus (EBV) in epithelial oral squamous cell carcinoma (OSCC), but an association of EBV with oral carcinogenesis has not yet been established. Although epigenetic alterations, such as aberrant DNA methylation, are known to contribute to the pathogenesis of oral cancer, the relationship of such alterations with EBV infection is little known. This study aimed to investigate the association between EBV infection and promoter methylation patterns of tumor-associated genes in OSCC tissues. A total of 165 of formalin-fixed paraffin-embedded OSCC tissues were studied (68 of EBV positive and 97 of EBV negative). The promoter methylation patterns were investigated for four tumor-associated genes, E-cadherin, p16 INK4a , p14 ARF , and MGMT, by using methylation-specific polymerase chain reaction (MSP). The frequencies of gene promoter hypermethylation in all cases were 47.3% for E-cadherin, 92.7% for p16 INK4a , 74.5% for p14 ARF , and 35.8% for MGMT. Interestingly, most of the analyzed gene promoters were more frequently hypermethylated in EBV-positive than EBV-negative cases, in particular the E-cadherin (56/22) and MGMT (38/21) gene promoters (p<0.05). Concomitantly, hypermethylation of multiple gene promoters (≥3) was encountered more frequently in EBV-positive samples. Hypermethylation of the E-cadherin promoter associated with EBV was more frequently observed in moderately and poorly differentiated OSCC tissues. These results indicate that epigenetic changes frequently occur in OSCCs and may partly be induced by EBV infection, therefore, EBV may involve in development and progression of the OSCCs.

Chidamide alleviates TGF-β-induced epithelial-mesenchymal transition in lung cancer cell lines.

Transforming growth factor-β (TGF-β)-induced epithelial-mesenchymal transition is a critical process in the initiation of metastasis of various types of cancer. Chidamide is a class I histone deacetylase inhibitor with anti-tumor activity. This study investigated the effects of chidamide on TGF-β-mediated suppression of E-cadherin expression in adenocarcinomic lung epithelial cells and the molecular mechanisms involved in these effects. Western blot analysis, confocal microscopy, Quantitative methyl-specific PCR and bisulfite sequencing were used to evaluate the effects of different treatments on chidamide ameliorating TGF-β induced-E-cadherin loss. H3 acetylation binding to the promoter of E-cadherin was detected by chromatin immunoprecipitations (CHIP). We found that chidamide reduced the level of lung cancer cell migration observed using a Boyden chamber assay (as an indicator of metastatic potential). Chidamide inhibited TGF-β-induced SMAD2 phosphorylation and attenuated TGF-β-induced loss of E-cadherin expression in lung cancer cells by Western blotting and confocal microscopy, respectively. Quantitative methyl-specific PCR and bisulfite sequencing revealed that TGF-β-enhanced E-cadherin promoter methylation was ameliorated in cells treated with chidamide. We demonstrated that histone H3 deacetylation within the E-cadherin promoter was required for TGF-β-induced E-cadherin loss; cell treatment with chidamide increased the H3 acetylation detected by CHIP. Taken together, our results demonstrate that TGF-β suppressed E-cadherin expression by regulating promoter methylation and histone H3 acetylation. Chidamide significantly enhanced E-cadherin expression in TGF-β-treated cells and inhibited lung cancer cell migration. These findings indicate that chidamide has a potential therapeutic use due to its capacity to prevent cancer cell metastasis.

Transcriptional Suppression of E-Cadherin by HPV-16 E6 and E7 Oncogenes is Independent of Hypermethylation of E-Cadherin Promoter

Background: Cervical cancer is one of the most common cancers observed in women worldwide, and its development is related to E6 and E7 two viral oncoproteins of high-risk human papillomavirus (HPV) types. Aberrant expression of E-cadherin, which is associated with epithelial-to-mesenchymal transition (EMT), is frequently observed in cervical cancer. Objectives: The mechanisms underlying E-cadherin suppression in cervical cancer are not clear; therefore, this experimental study from Iran was designed to elucidate the relationship of DNA methyltransferase expression and E-cadherin promoter methylation with E-cadherin expression in HPV-16 E6- and E7-expressing cells. Materials and Methods: Real-time PCR and western blot were used to determine the effects of HPV-16 E6 and E7 on E-cadherin, DNMT1, DNMT3a, and DNMT3b expression in HCT-116 cell line. We also analyzed E-cadherin promoter methylation in cells expressing HPV-16 E6 and E7 oncoproteins by bisulfite sequencing. Results: HPV-16 E6 and E7 proteins reduced E-cadherin expression 3.7 and 2.2 times when compared with control cells (P = 0.0221 and P = 0.0461, respectively). This reduction was greater in HPV-16 E6-expressing cells than in HPV-16 E7-expressing cells. Although HPV-16 E6 and E7 increased DNA methyltransferase 1 expression 2.6 and 3.4 times, respectively (P = 0.0133 and P = 0.0113) when compared with control cells, they was no E-cadherin promoter methylation. Conclusions: Unlike other cancer-associated viruses (HBV, HCV, and EBV), reduction in E-cadherin expression in HPV-16 E6- and E7-expressing cells is not due to hypermethylation of the E-cadherin promoter.

E-cadherin Promoter Methylation Comparison and Correlation with the Pathological Features of the Squamous Cell Carcinoma of Esophagus in the High Risk Region

E-cadherin is among tumor suppressor genes which mostly subjects to the down-regulation in squamous cell carcinoma of esophagus (SCCE). The gene is tightly associated with the tumor invasion and metastasis in multiple human cancers, especially SCCE. CpG islands’ methylation in the promoter region of E-cadherin is among the mechanisms that have been suggested for the E-cadherin silencing, however, little is known regarding (SCCE) in the high risk region of the world. To establish a correlation between E-Cadherin promoter methylation with the pathological features of SCCE, as well as history, and demographic features we undertook the present investigation. Following to the surgical resection tissue samples of the 44 patients with SCCE was used for E-cadherin promoter methylation examination. Analysis was done using methylation specific polymerase chain reaction (MS-PCR). Furthermore, for evaluating E-daherin expression levels, reverse transcription PCR (RT-PCR) was applied.Results have indicated that twenty four out of the forty four tumor DNA samples (54.5%) were aberrantly methylated. In contrast, all normal tissues were found to be unmethylated. In addition a significant association was found between methylation status of E-Cadherin promoter with type I and II of the tumor differentiation (p=0.024), stages T2 and T3 of tumors (p= 0.026), as well as lymph node invasion (p= 0.004). E-cadherin tumor suppressor gene subjects to epigenetic silencing through aberrant promoter CpG islands methylation; a mechanism which is most commonly contributed with the other important tumor suppressor genes in the SCCE carcinogenesis in the world’s highest risk region for SCCE.

Role of hMLH1 and E-Cadherin Promoter Methylation in Gastric Cancer Progression

Gastric cancer (GC) is one of the leading causes of cancer-related death in Iran. Genome stability is one of the main genetic issues in cancer biology which is governed via the different repair systems such as DNA mismatch repair (MMR). A clear correlation between MMR defects and tumor progression has been shown. Beside the genetic mutations, epigenetic changes also have a noticeable role in MMR defects. Here, we assessed promoter methylation status and the level of hMLH1mRNA expression as the main component of MMR system in 51 GC patients using the methylation-specific PCR and real-time PCR, respectively. Moreover, we performed a promoter methylation study of the E-cadherin gene promoter. It was observed that, 12 out of 39 cases (23.5%) had hMLH1 overexpression. Hypermethylation of hMLH1 and E-cadherin promoter regions were observed in 25.5 and 36.4%, respectively. Although, there was no significant correlation between hMLH1 mRNA expression and clinicopathological features, there are significant correlations between E-cadherin promoter methylation and tumor stage (p = 0.028) and location (p = 0.025). The rate of hMLH1 promoter methylation in this study was lower than that in the other population, showing the importance of the other mechanisms, in gastric tumorigenesis. The results of this study indicate that DNA repair system is adversely affected by hypermethylation of hMLH1 in a fraction of gastric cancer patients. Additionally, E-cadherin hypermethylation seen in a subset of our gastric cancer patients is consistent with other reports showing correlation with aggressiveness and metastasis of gastric cancer.

DNA methyltransferase inhibitor assay system based on the HBx-induced DNA methylation of E-cadherin

We here report a simple assay system for DNA methyltransferase (DNMT) inhibitors based on the HBx-induced DNA methylation of E-cadherin. A stable cell line named G1 was generated by co-transfecting E-cadherin luciferase reporter and HBx-expression plasmid into HepG2 cells. Treatment of G1 cells with DNMT inhibitors, 5-azacytidine, 5-aza-2′-deoxycytidine, and procainamaid, dose-dependently inhibited DNA methylation of E-cadherin promoter in the reporter, resulting in up-regulation of luciferase levels and its enzyme activity. Treatment with all-trans retinoic acid that is known to inhibit DNMT expression, also induced similar effects. Our system can be useful for development of epi-drugs targeting DNA methylation in malignancies.

Meta-analysis of association between E-cadherin promoter methylation and lung cancer risk

背景与目的上皮细胞钙粘蛋白E-cadherin在维持上皮的极性和结构完整性等方面起着重要作用，并与肿瘤的发生、浸润和转移密切相关。E-cadherin基因启动子区甲基化与肿瘤发生发展的关系一直是研究的热点，而该基因甲基化与肺癌易感性的关系尚存在争议。本研究旨在通过meta分析的方法更好地探讨E-cadherin基因启动子区甲基化与肺癌易感性之间的关系。方法利用PubMed/MedLine和EMBASE数据库检索2013年3月以前的相关文献，由两位研究人员做独立文献筛选和资料提取，并交叉审核。使用优势比（odds ratio, OR）及95%置信区间（confidence interval, CI）衡量E-cadherin基因启动子区甲基化与肺癌易感性关系的强弱。结果根据检索条件，共有13项研究（共涉及1, 288例实验样本）被纳入当前的meta分析。分析结果显示E-cadherin基因启动子区甲基化明显增加了肺癌发生风险（OR=4.04, 95%CI: 2.00-8.13, P < 0.001），且这种相关性同时存在于亚洲人群和高加索人群（OR=3.28, 95%CI: 1.20-8.92; OR=5.72, 95%CI: 2.40-13.62）。结论E-cadherin基因启动子区甲基化与肺癌易感性之间存在明显相关性。

Altered expression of β-catenin, E-cadherin, and E-cadherin promoter methylation in epithelial ovarian carcinoma

The aim of the study was to evaluate the immunoexpression of E-cadherin, β-catenin, and Ki-67, as well as the promoter methylation of E-cadherin gene in epithelial ovarian cancer (EOC), as well as to find a possible relationship between the immunoexpression and hypermethylation. Promoter methylation was studied using methylation-specific PCR in 86 malignant cases, 14 low malignant potential (LMP) tumors and 19 benign cystadenomas. Immunohistochemical expression was carried out in 64 malignant cases, 8 LMP tumors, and 11 benign cystadenomas. Immunoexpression of E-cadherin was reduced in EOC, while 100 % expression was seen in LMP tumors and benign cystadenomas. An interesting observation was the nuclear expression of E-cadherin in a high percentage of cancers, which showed a positive correlation with Ki-67. Β-Catenin expression showed heterogeneous localization with increased nuclear localization, which was significantly higher in cases that did not express E-cadherin. Promoter methylation of E-cadherin was 36, 14, and 11 % in EOC, LMP tumors, and benign cystadenomas, respectively. Our results suggest that reduced expression of E-cadherin is associated with promoter methylation of E-cadherin gene, in addition to providing evidence for the aberrant nuclear localization of E-cadherin in EOC.

Exposures in early life: associations with DNA promoter methylation in breast tumors.

There is evidence that epigenetic changes occur early in breast carcinogenesis. We hypothesized that early-life exposures associated with breast cancer would be associated with epigenetic alterations in breast tumors. In particular, we examined DNA methylation patterns in breast tumors in association with several early-life exposures in a population-based case-control study. Promoter methylation of E-cadherin, p16 and RAR-β2 genes was assessed in archived tumor blocks from 803 cases with real-time methylation-specific PCR. Unconditional logistic regression was used for case-case comparisons of those with and without promoter methylation. We found no differences in the prevalence of DNA methylation of the individual genes by age at menarche, age at first live birth and weight at age 20. In case-case comparisons of premenopausal breast cancer, lower birth weight was associated with increased likelihood of E-cadherin promoter methylation (OR = 2.79, 95% CI, 1.15-6.82, for ⩽2.5 v. 2.6-2.9 kg); higher adult height with RAR-β2 methylation (OR = 3.34, 95% CI, 1.19-9.39, for ⩾1.65 v. <1.60 m); and not having been breastfed with p16 methylation (OR = 2.75, 95% CI, 1.14-6.62). Among postmenopausal breast cancers, birth order was associated with increased likelihood of p16 promoter methylation. Being other than first in the birth order was inversely associated with likelihood of ⩾1 of the three genes being methylated for premenopausal breast cancers, but positively associated with methylation in postmenopausal women. These results suggest that there may be alterations in methylation associated with early-life exposures that persist into adulthood and affect breast cancer risk.

Molecular Staging of Surgical Margins in Oral Squamous Cell Carcinoma Using Promoter Methylation of p16INK4A, Cytoglobin, E-cadherin, and TMEFF2

Local recurrence in oral squamous cell carcinoma (OSCC) despite clear surgical margins may indicate the presence of residual, sub-microscopic disease. Molecular assessment of surgical margins may provide a greater prognostic sensitivity compared to histopathology. We aimed to determine whether promoter methylation in deep and mucosal resection margins can predict recurrence in OSCC. Forty-eight consecutive OSCC cases were recruited and a 5mm(3) tumor sample plus 5 deep and 5 mucosal margin samples were snap frozen. Clinical, pathological, adjuvant therapy, and outcome data were recorded. Tumors were informative if >5% promoter methylation was found for ≥1 of 4 genes using qMSP. Margins were declared molecularly positive if >1% promoter methylation was found in any margin. Thirty (63%) of 48 cases were methylation informative. Mucosal margin samples were largely positive for methylation (26 of 30, 87%), indicating the presence of field cancerization. Methylation at ≥1 gene promoters in ≥1 deep margin correlated with the presence of close/involved mucosal margins (P=0.027) and increased pT status (P=0.027) but not the status of deep margins, recurrence, or survival. The current gene panel did not add prognostic information to histopathological reporting of resection margins. Future efforts should concentrate on improving gene selection, informativity, and assay performance in the patient group with intermediate indications for adjuvant therapy.

Epigenetic inactivation of the E-cadherin gene in eyelid sebaceous gland carcinoma

E-cadherin and β-catenin are crucial components of the cell-cell adhesion complex. Their loss has often been associated with tumour metastasis and poor clinical outcome. Both loss of E-cadherin at the cell membrane and a stabilizing mutation in CTNNB1 (β-catenin gene) have been associated with ovarian, colorectal, hepatocellular and nonmelanoma skin cancer, such as squamous and basal cell carcinomas. Absence of E-cadherin may be caused by promoter hypermethylation of the E-cadherin gene (CDH1). To determine the role of E-cadherin promoter hypermethylation and CTNNB1 gene mutation in the aggressive behaviour of sebaceous gland carcinoma of the eyelid. Thirty-six cases of sebaceous gland carcinoma were subjected to E-cadherin methylation-specific polymerase chain reaction and mutational analysis for the CTNNB1 gene. E-cadherin and β-catenin staining was evaluated by immunohistochemistry. Results were correlated with the clinicopathological features of sebaceous gland carcinoma. nMethylation of the E-cadherin promoter region was detected in 72% of eyelid sebaceous gland carcinoma cases and loss of E-cadherin immunostaining in 83%. E-cadherin promoter hypermethylation showed a significant association with the loss of membranous E-cadherin (P = 0·038) and it was of borderline significance with reduced disease-free survival (P = 0·05). It was also found to be associated with advanced age (73%), tumour size ≥ 2 cm (77%), orbital invasion (83%), lymph node metastasis (60%), tumour recurrence (60%) and poor histological differentiation (90%). DNA sequencing revealed no stabilizing β-catenin gene mutation in sebaceous gland carcinoma. Loss of membranous β-catenin was observed in 61% cases, which associated significantly with both E-cadherin promoter methylation (P = 0·0262) and loss of E-cadherin membranous localization (P=0·0015). Epigenetic inactivation of the E-cadherin gene causes loss of membrane-bound E-cadherin and could contribute to the reduced disease-free survival in eyelid sebaceous gland carcinoma. Mutations in the β-catenin gene do not seem to be involved in the pathogenesis of eyelid sebaceous gland carcinoma.

Abstract 4077: Chemosensitizing effects of the novel, small molecule DNA methylation inhibitor SGI-110 in ovarian cancer

Abstract Deoxycytosine methylation of CpG islands in promoter regions of tumor suppressor genes (TSGs) plays a prominent role in the development and progression of drug-resistant epithelial ovarian cancer (OC). We previously demonstrated in a phase I/II trial that the DNA methylation inhibitor decitabine alters DNA methylation and restores platinum sensitivity in platinum-resistant OC patients, resulting in significant clinical activity. SGI-110 (Astex Pharmaceuticals) is a DNA hypomethylating agent with demonstrated activity in restoring silenced TSG expression in cancer cells by reversal of DNA methylation. As a decitabine-deoxyguanosine dinucleotide, SGI-110 has been shown to be less prone to deamination by cytidine deaminase and could have advantages over decitabine, such as better stability, less toxicity and a more convenient and less frequent SQ administration. We examined SGI-110 for its ability to inhibit OC cell proliferation and to demethylate and induce TSGs in vitro and in vivo. A 48 hour pretreatment with SGI-110 resensitized drug-resistant ovarian cancer cell lines to cisplatin (3-fold reduction in IC50 of cisplatin). In A2780 platinum sensitive OC cells, SGI-110 pre-treatment for 5 days reduced cisplatin IC50 by &amp;gt;2-fold. Prolonged (7 days) SGI-110 treatment reduced by 2-fold the number of ALDH1+ cells in SKOV3 and A2780 cells, suggesting an effect on the stem cell population, potentially involved in drug resistance. SGI-110 induced significant demethylation of TSGs ras-associated domain family 1A (RASSF1A) and human MutL homologue-1 (MLH1) and the differentiation-associated gene HOXA10; furthermore, RASSF1A, MLH1 and HOXA10 gene reexpression was also observed after 48 hour SGI-110 treatment. SGI-110 prevented TGF-β induced epithelial to mesenchymal transition of OC cells, as measured morphologically and by quantification of E-cadherin, Zeb1, Slug and miR200c expression levels. SGI-110 suppressed E-cadherin promoter methylation induced by TGF-β. We then examined the in vivo tolerability and efficacy of SGI-110, singly and in combination with cisplatin, in nude athymic mice. Mice were injected qd or bi-weekly with SGI-110 (sc) and/or cisplatin (ip) and body weights were measured twice weekly. In the qd regimen, SGI-110 2 mg/kg plus 4 mg/kg cisplatin was tolerated. The 10 mg/kg bi-weekly dose of SGI-110 was tolerated, either alone or in combination with 2 mg/kg cisplatin (5mg/kg SGI-110 plus 4 mg/kg cisplatin was similarly tolerated). SGI-110 induced significant global hypomethylation, as determined by pyrosequencing analysis of LINE1 demethylation in peripheral blood mononuclear cells. We are currently assessing the activity of SGI-110 in combination with cisplatin to retard the growth of platinum resistant human ovarian cancer xenografts. In summary, SGI-110 combined with platinum is a promising clinical combination for the therapy of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4077. doi:1538-7445.AM2012-4077

Promoter Methylation of E-Cadherin, p16, and RAR-β2 Genes in Breast Tumors and Dietary Intake of Nutrients Important in One-Carbon Metabolism

Aberrant DNA methylation plays a critical role in carcinogenesis, and the availability of dietary factors involved in 1-carbon metabolism may contribute to aberrant DNA methylation. We investigated the association of intake of folate, vitamins B2, B6, B12, and methionine with promoter methylation of E-cadherin, p16, and RAR-β2 genes in archived tumor tissues from incident, primary breast cancer cases in a population-based case-control study. Real-time methylation-specific PCR was performed on 803 paraffin-embedded samples; usual dietary intake was queried from a food frequency questionnaire. Unconditional logistic regression was used to derive adjusted odds ratios and 95% confidence intervals for likelihood of promoter methylation for high compared to low intake of those 1-carbon nutrients. Overall, in case-case comparisons, dietary intakes of folate, vitamins B2, B6, B12, and methionine were not associated with likelihood of promoter methylation of E- cadherin, p16, and RAR-β2 for all cases combined or within strata defined by menopausal status and estrogen receptor status in this study. This finding, however, does not exclude the possibility that intake of such nutrients might have the ability to modulate promoter methylation in normal or premalignant (dysplastic) breast tissue.

Body mass and DNA promoter methylation in breast tumors in the Western New York Exposures and Breast Cancer Study

The mechanism of the observed association between body mass, particularly centralized body fat, and postmenopausal breast cancer risk is not well understood. We hypothesized that body mass may affect DNA methylation through increased estrogen and chronic inflammation. The association between body mass and promoter methylation in breast tumors was investigated in a population-based, case-control study. The promoter methylation of E-cadherin, p16, and RAR-β(2) genes was assessed in breast tumor blocks from 803 pre- and postmenopausal cases by using real-time methylation-specific polymerase chain reaction. Unconditional logistic regression was used to derive the adjusted OR and 95% CI for case-case comparisons of tumors with and without promoter methylation of the genes. The frequency of promoter methylation was 20% for E-cadherin, 25.9% for p16, and 27.5% for RAR-β(2). There was no difference in the prevalence of the DNA methylation of individual genes by BMI, waist-to-hip ratio (WHR), or lifetime weight change between the age of 20 y and the present. However, in a case-case comparison of postmenopausal breast cancer, a greater WHR was associated with an increased likelihood of ≥1 of the 3 genes being methylated (OR: 1.85; 95% CI: 1.10, 3.11; P-trend < 0.02). We showed that WHR was associated with DNA promoter methylation of ≥1 of 3 genes in postmenopausal breast tumors. It may be that the association of body fat composition and postmenopausal breast cancer is related to altered DNA methylation. However, future studies in other populations and with an examination of the methylation of more genes are needed.

Abstract 5610: Air pollution exposure and promoter methylation of E-cadherin, p16, and RAR-β in breast cancer tumors from cases in the WEB study

Abstract Purpose: Aberrant methylation has been shown to be associated with breast cancer tumor tissue samples and ambient air pollution exposure has been shown to be associated with aberrant methylation of gene promoter regions; we investigated potential associations between air pollution exposure in early life and promoter methylation of genes among breast cancer tumors. Methods: Paraffin embedded, formalin fixed tumor tissue blocks were obtained for 803 breast cancer cases participating in the WEB study and assayed for DNA methylation status of three promoters regions of E-cadherin, p16 and RAR-β. Methylation specific PCR was used to determine methylation status. Historical annual ambient total suspended particulate matter (TSP) concentration (μg/m3) was collected from 87 air monitoring sites in Erie and Niagara Counties dating as far back as the late 1950's. Residential addresses were obtained for the time of birth, time of menarche, time of first birth if applicable, and 10 years prior to diagnosis with breast cancer. Using Arc View software, the addresses were geocoded and overlaid with the air pollution information to estimate exposure to TSP at each time point. TSP exposures varied greatly over time, therefore they were categorized into high and low based on the mean exposure among the non-breast cancer controls in the WEB study. This analysis consists of case-case comparisons, comparing TSP exposure to DNA methylation status among breast cancer patients. Odds ratios and 95% confidence limits were calculated with unconditional logistic regression, adjusting for potential confounders. We stratified all analyses by menopausal status. Results: Among pre- but not postmenopausal women, there was an increased likelihood of a case having methylation of the p16 promoter region for those exposed to &amp;gt;109.03 μg/m3 of TSP at first birth compared to those exposed to ≤109.03 μg/m3 (n=123; OR=6.5, 95% CI: 1.2-34. 1). E-cadherin promoter region methylation was inversely associated with high TSP at birth (&amp;gt; 137.88 μg/m3) among the pre-menopausal women (n=138; OR=0.2, 95% CI: 0.1-0.8) but not for post-menopausal women (n=289; OR=0.8, 95% CI: 0.4-1.6) nor for the other genes. There was no evidence of association between TSP exposures and RAR-β promoter region methylation for any of the time periods. Conclusion: Air pollution exposure at birth was much higher than exposure at later times due to increasing regulation. Exposure to higher levels of TSP at birth was associated with decreased likelihood of promoter region methylation for E-cadherin, particularly for premenopausal cases. TSP exposure at the time of a first birth was associated with increased likelihood of p16 promoter methylation in pre- but not postmenopausal breast tumors. While our results are necessarily preliminary, they suggest that exposure to air pollutants in early life may affect promoter methylation of E-cadherin and p16 genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5610. doi:10.1158/1538-7445.AM2011-5610