Quantitative analysis of promoter methylation in exfoliated epithelial cells isolated from breast milk of healthy women

Promoter methylation analysis of genes frequently silenced in breast cancer is a promising indicator of breast cancer risk, as these methylation events are thought to occur long before presentation of disease. The numerous exfoliated epithelial cells present in breast milk may provide the breast epithelial DNA needed for detailed methylation analysis and assessment of breast cancer risk. Fresh breast milk samples and health, lifestyle, and reproductive history questionnaires were collected from 111 women. Pyrosequencing analysis was conducted on DNA isolated from the exfoliated epithelial cells immunomagnetically separated from the total cell population in the breast milk of 102 women. A total of 65 CpG sites were examined in six tumor suppressor genes: PYCARD (also known as ASC or TMS1), CDH1, GSTP1, RBP1 (also known as CRBP1), SFRP1, and RASSF1. A sufficient quantity of DNA was obtained for meaningful analysis of promoter methylation; women donated an average of 86 ml of milk with a mean yield of 32,700 epithelial cells per ml. Methylation scores were in general low as expected of benign tissue, but analysis of outlier methylation scores revealed a significant relationship between breast cancer risk, as indicated by previous biopsy, and methylation score for several CpG sites in CDH1, GSTP1, SFRP1, and RBP1. Methylation of RASSF1 was positively correlated with women’s age irrespective of her reproductive history. Promoter methylation patterns in DNA from breast milk epithelial cells can likely be used to assess breast cancer risk. Additional studies of women at high breast cancer risk are warranted.     

Quantitative analysis of promoter methylation in exfoliated epithelial cells isolated from breast milk of healthy women

Promoter methylation analysis of genes frequently silenced in breast cancer is a promising indicator of breast cancer risk, as these methylation events are thought to occur long before presentation of disease. The numerous exfoliated epithelial cells present in breast milk may provide the breast epithelial DNA needed for detailed methylation analysis and assessment of breast cancer risk. Fresh breast milk samples and health, lifestyle and reproductive history questionnaires were collected from 111 women. Pyrosequencing analysis was conducted on DNA isolated from the exfoliated epithelial cells immunomagnetically separated from the total cell population in the breast milk of 102 women. A total of 65 CpG sites were examined in six tumor suppressor genes: PYCARD (also known as ASC or TMS1), CDH1, GSTP1, RBP1 (also known as CRBP1), SFRP1 and RASSF1. A sufficient quantity of DNA was obtained for meaningful analysis of promoter methylation; women donated an average of 86 ml of milk with a mean yield of 32,700 epithelial cells per ml. Methylation scores were in general low as expected of benign tissue, but analysis of outlier methylation scores revealed a significant relationship between breast cancer risk, as indicated by previous biopsy and methylation score, for several CpG sites in CDH1, GSTP1, SFRP1 and RBP1. Methylation of RASS F1 was positively correlated with women''s age irrespective of her reproductive history. Promoter methylation patterns in DNA from breast milk epithelial cells can likely be used to assess breast cancer risk. Additional studies of women at high breast cancer risk are warranted.Key words: biomarker, pyrosequencing, promoter methylation, breast epithelial cells, breast milk, breast cancer risk, parity, age-related promoter methylation, pregnancy-associated protection from breast cancer     

Detection of promoter methylation of tumor suppressor genes in serum DNA of breast cancer cases and benign breast disease controls

Tumors are capable of shedding DNA into the blood stream. This shed DNA may be recovered from serum or plasma. The objective of this study was to evaluate whether pyrosequencing promoter DNA in a panel of 12 breast cancer-related genes (APC, BRCA1, CCND2, CDH1, ESR1, GSTP1, HIN1, P16, RARβ, RASSF1, SFRP1 and TWIST) to measure the degree of methylation would lead to a useful serum-based marker of breast cancer. Serum was obtained from women who were about to undergo a breast biopsy or mastectomy at three hospitals from 1977 to 1987 in Grand Rapids, MI USA. We compared the methylation status of 12 genes in serum DNA obtained from three groups of postmenopausal women (mean age at blood collection: 63.0 y; SD 9.9; range 35–91): breast cancer cases with lymph node-positive disease (n = 241); breast cancer cases with lymph node-negative disease (n = 63); and benign breast disease control subjects (n = 234). Overall, median levels of promoter methylation were low, typically below 5%, for all genes in all study groups. For all genes, median levels of methylation were higher (by 3.3 to 47.6%) in lymph node-positive breast cancer cases than in the controls. Comparing mean methylation level between lymph-node positive cases and controls, the most statistically significant findings, after adjustment of the false-positive rate (q-value), were for TWIST (p = 0.04), SFRP1 (p = 0.16), ESR1 (p = 0.17), P16 (p = 0.19) and APC (p = 0.19). For two of these four genes (TWIST, P16), the median methylation level was also highest in lymph-node positive cases, intermediate in lymph node-negative cases and lowest in the controls. The percent of study subjects with mean methylation scores ≥ 5% was higher among lymph node-positive cases than controls for ten genes, and significantly higher for HIN1 and TWIST (22.0 vs. 12.2%, p = 0.04 and 37.9 vs. 24.5%, p = 0.004, respectively). Despite relatively consistent variation in methylation patterns among groups, these modest differences did not provide sufficient ability to distinguish between cases and controls in a clinical setting.     

Detection of promoter methylation of tumor suppressor genes in serum DNA of breast cancer cases and benign breast disease controls

Tumors are capable of shedding DNA into the blood stream. This shed DNA may be recovered from serum or plasma. The objective of this study was to evaluate whether pyrosequencing promoter DNA in a panel of 12 breast cancer-related genes (APC, BRCA1, CCND2, CDH1, ESR1, GSTP1, HIN1, P16, RARβ, RASSF1, SFRP1 and TWIST) to measure the degree of methylation would lead to a useful serum-based marker of breast cancer. Serum was obtained from women who were about to undergo a breast biopsy or mastectomy at three hospitals from 1977 to 1987 in Grand Rapids, MI USA. We compared the methylation status of 12 genes in serum DNA obtained from three groups of postmenopausal women (mean age at blood collection: 63.0 y; SD 9.9; range 35–91): breast cancer cases with lymph node-positive disease (n = 241); breast cancer cases with lymph node-negative disease (n = 63); and benign breast disease control subjects (n = 234). Overall, median levels of promoter methylation were low, typically below 5%, for all genes in all study groups. For all genes, median levels of methylation were higher (by 3.3 to 47.6%) in lymph node-positive breast cancer cases than in the controls. Comparing mean methylation level between lymph-node positive cases and controls, the most statistically significant findings, after adjustment of the false-positive rate (q-value), were for TWIST (p = 0.04), SFRP1 (p = 0.16), ESR1 (p = 0.17), P16 (p = 0.19) and APC (p = 0.19). For two of these four genes (TWIST, P16), the median methylation level was also highest in lymph-node positive cases, intermediate in lymph node-negative cases and lowest in the controls. The percent of study subjects with mean methylation scores ≥ 5% was higher among lymph node-positive cases than controls for ten genes, and significantly higher for HIN1 and TWIST (22.0 vs. 12.2%, p = 0.04 and 37.9 vs. 24.5%, p = 0.004, respectively). Despite relatively consistent variation in methylation patterns among groups, these modest differences did not provide sufficient ability to distinguish between cases and controls in a clinical setting.     

Relationship between tumor DNA methylation status and patient characteristics in African-American and European-American women with breast cancer

Aberrant DNA methylation is critical for development and progression of breast cancer. We investigated the association of CpG island methylation in candidate genes and clinicopathological features in 65 African-American (AA) and European-American (EA) breast cancer patients. Quantitative methylation analysis was carried out on bisulfite modified genomic DNA and sequencing (pyrosequencing) for promoter CpG islands of p16, ESR1, RASSF1A, RARβ2, CDH13, HIN1, SFRP1 genes and the LINE1 repetitive element using matched paired non-cancerous and breast tumor specimen (32 AA and 33 EA women). Five of the genes, all known tumor suppressor genes (RASSF1A, RARβ2, CDH13, HIN1 and SFRP1), were found to be frequently hypermethylated in breast tumor tissues but not in the adjacent non-cancerous tissues. Significant differences in the CDH13 methylation status were observed by comparing DNA methylation between AA and EA patients, with more obvious CDH13 methylation differences between the two patient groups in the ER- disease and among young patients (age<50). In addition, we observed associations between CDH13, SFRP1, and RASSF1A methylation and breast cancer subtypes and between SFRP1 methylation and patient's age. Furthermore, tumors that received neoadjuvant therapy tended to have reduced RASSF1A methylation when compared with chemotherapy na?ve tumors. Finally, Kaplan Meier survival analysis showed a significant association between methylation at 3 loci (RASSF1A, RARβ2 and CDH13) and reduced overall disease survival. In conclusion, the DNA methylation status of breast tumors was found to be significantly associated with clinicopathological features and race/ethnicity of the patients.     

Global and gene-specific promoter methylation analysis in primary hyperparathyroidism

Epigenetic mechanisms involved in primary hyperparathyroidism are poorly understood as studies are limited. In order to understand the role of aberrant DNA promoter methylation in the pathogenesis of parathyroid tumors, we have quantified the CpG island promoter methylation density of several candidate genes including APC (promoter 1A and 1B), β-catenin (CTNNB1), CASR, CDC73/HRPT2, MEN1, P16 (CDKN2A), PAX1, RASSF1A, SFRP1 and VDR in 72 parathyroid tumors and 3 normal parathyroid references using bisulfite pyrosequencing. Global methylation levels were assessed for LINE-1. We also compared methylation levels with gene expression levels measured by qRT-PCR for genes showing frequent hypermethylation. The adenomas displayed frequent hypermethylation of APC 1A (37/66; 56%), RASSF1A (34/66; 52%) and β-catenin (19/66; 29%). One of the three atypical adenomas was hypermethylated for APC 1A. The three carcinomas were hypermethylated for RASSF1A and SFRP1, and the latter was only observed in this subtype. The global methylation density was similar in tumors (mean 70%) and parathyroid reference samples (mean 70%). In general, hypermethylated genes had reduced expression in the parathyroid adenomas using qRT-PCR. Among the adenomas, methylation of APC 1A correlated with adenoma weight (r = 0.306, p < 0.05). Furthermore, the methylation status of RASSF1A correlated with each of APC 1A (r = 0.289, p < 0.05) and β-catenin (r = 0.315, p < 0.01). Our findings suggest a role for aberrant DNA promoter methylation of APC 1A, β-catenin and RASSF1A in a subset of parathyroid tumors.     

DNA methylation in pre-diagnostic serum samples of breast cancer cases: Results of a nested case–control study

Background: Promoter methylation of tumor suppressor genes is a frequent and early event in breast carcinogenesis. Paired tumor tissue and serum samples from women with breast cancer show that promoter methylation is detectable in both sample types, with good concordance. This suggests the potential for these serum markers to be used for breast cancer detection. Methods: The current study was a case–control study nested within the prospective New York University Women's Health Study cohort aimed to assess the ability of promoter methylation in serum to detect pre-clinical disease. Cases were women with blood samples collected within the 6 months preceding breast cancer diagnosis (n = 50). Each case was matched to 2 healthy cancer-free controls and 1 cancer-free control with a history of benign breast disease (BBD). Results: Promoter methylation analysis of four cancer-related genes: — RASSF1A, GSTP1, APC and RARβ2, — was conducted using quantitative methylation-specific PCR. Results showed that the frequency of methylation was lower than expected among cases and higher than expected among controls. Methylation was detected in the promoter region of: RASSF1A in 22.0%, 22.9% and 17.2% of cases, BBD controls and healthy controls respectively; GSTP1 in 4%, 10.4% and 7.1% respectively; APC in 2.0%, 4.4% and 4.2% respectively and RARβ2 in 6.7%, 2.3% and 1.1% respectively. Conclusion: Methylation status of the four genes included in this study was unable to distinguish between cases and either control group. This study highlights some methodological issues to be addressed in planning prospective studies to evaluate methylation markers as diagnostic biomarkers.     

Methylation profiling of urothelial carcinoma in bladder biopsy and urine

OBJECTIVE: To test DNA methylation profiling in detection of urothelial carcinoma in urine. STUDY DESIGN: Thirty-three bladder specimens were analyzed for the DNA p16INK4a, RASSF1, APC, GSTP, E-Cad and CyclinD2 genes to determine if there is a difference in gene methylation between benign and malignant cases. Urine samples were analyzed in a feasibility study. Finally, methylation profiles of urine samples were obtained and compared with follow-up biopsy diagnoses. RESULTS: We found methylated genes in 18% benign, 37% urothelial carcinoma in situ and 93% infiltrating urothelial carcinoma cases (p = 0.001). Methylation profiles from the 18 urine samples revealed a significantly higher prevalence of methylated genes in carcinoma cases than benign cases (100% vs. 50%, p = 0.025). We analyzed methylation profiles in 37 cytologically atypical urine samples with malignant or benign diagnosis on surgical follow-up andfound that only APC (55% in malignant vs. 0% in benign, p=0.025) and CyclinD2 were differentially methylated (35% in malignant vs. 0% in benign, p=0.2) while p14ARF, p16INK4a, RASSF1, GSTP and E-Cad had similar methylation profiles. CONCLUSION: These results suggest that methylation of p14ARF, p16INK4a, RASSF1, GSTP and E-Cad genes may not accurately identify carcinoma, but methylated APC and CyclinD2 might be useful biomarkers for urothelial carcinoma in urine.     

Methylation status and protein expression of RASSF1A in breast cancer patients

Recently genetics and epigenetics alterations have been found to be characteristic of malignancy and hence can be used as targets for detection of neoplasia. RAS association domain family protein 1A (RASSF1A) gene hypermethylation has been a subject of interest in recent researches on cancer breast patients. The aim of the present study was to evaluate whether RASSF1A methylation status and RASSF1A protein expression are associated with the major clinico-pathological parameters. One hundred and twenty breast cancer Egyptian patients and 100-control subjects diagnosed with benign lesions of the breast were enrolled in this study. We evaluated RASSF1A methylation status in tissue and serum samples using Methyl specific PCR together with RASSF1A protein expression in tissues by immunohistochemistry. Results were studied in relation to known prognostic clinicopathological features in breast cancer. Frequency of RASSF1A methylation in tissues and serum were 70 and 63.3 % respectively and RASSF1A protein expression showed frequency of 46.7 %. There was an association between RASSF1A methylation in tissues, serum and loss of protein expression in tissues with invasive carcinoma, advanced stage breast cancer, L.N. metastasis, ER/PR and HER2 negativity. RASSF1A methylation in serum showed high degree of concordance with methylation in tissues (Kappa = 0.851, P < 0.001). RASSF1A hypermethylation in tissues and serum and its protein expression may be a valid, reliable and sensitive tool for detection and follow up of breast cancer patients.     

Effect of aberrant promoter methylation of FHIT and RASSF1A genes on susceptibility to cervical cancer in a North Indian population

As current evidence suggests the involvement of epigenetic modification of tumour suppressor genes in human cancer, we investigated the aberrant promoter methylation of FHIT and RASSF1A genes in human papillomavirus (HPV)-mediated cervical cancer in Indian women. We analysed 60 cervical cancer tissue biopsies of different clinical stage and histological grading and 23 healthy control samples with normal cervical cytology. Methylation-specific polymerase chain reaction (MSP) was performed to analyse the methylation status of FHIT and RASSF1A genes and confirmed by sequencing. Both patients and controls were screened for HPV infection and 98% of the HPV-infected cases showed positivity for HPV type 16. Aberrant promoter methylation of the FHIT gene was found in 28.3% (17/60) of cases and of the RASSF1A gene in 35.0% (21/60) of cases; promoter methylation of both the genes was found in 13.3% (8/60) of cervical cancer cases. Methylation was significantly (p<0.01) associated with the cervical cancer cases compared with controls. None of the 23 controls was found to be methylated in either of these genes. This is the first study indicating a correlation between the promoter methylation of FHIT and RASSF1A genes and the clinical stage and histological grading of cervical carcinoma in Indian women. Future studies are underway to examine the practical implications of these findings for use as a biomarker.     

Epigenetic Modulations Induction Using DSCR1 Ectopic Expression in Breast Cancer Cells

Today, prognosis, diagnosis and treatment of cancers are progressing with non-invasive methods, including investigation and modification of the DNA methylation profile in cancer cells. One of the effective factors in regulating gene expression in mammals is DNA methylation. Methylation alterations of genes by external factors can change the expression of genes and inhibit the cancer. In the present study, we investigated the effect of Down syndrome critical region 1 gene (DSCR1) ectopic expression on the methylation status of the BCL-XL, ITGA6, TCF3, RASSF1A, DOK7, VIM and CXCR4 genes in breast cancer cell lines. The effect of DSCR1 ectopic expression on cell viability in MCF7, MDA-MB-468, MDA-MB-231 and MCF10A cell lines was evaluated using MTT assay after the cells treated by lentivirus vectors harboring DSCR1 for 72 hours. Methylation status of BCL-XL, ITGA6, TCF3, RASSF1A, DOK7, VIM and CXCR4 genes in breast cancer cell lines was assessed by Restriction Enzyme PCR (REP) method. Also, methylation changes of these genes in breast cancer cell lines after treatment by lentivirus vectors harboring DSCR1 for 7 days were analyzed by REP method. To confirm the effect of DSCR1 on methylation of genes, Real-time PCR was performed. The MTT assay results indicated that DSCR1 ectopic expression reduced cell viability in all three human breast cancer cell lines. Our results showed that DSCR1 ectopic expression after 6 days reversed the hypomethylation status of the BCL-XL, ITGA6, TCF3, VIM and CXCR4 genes and hypermethylation of RASSF1A and DOK7 genes. The expression levels of BCL-XL, ITGA6, TCF3, VIM and CXCR4 mRNA significantly reduced (P<0.05) and the expression levels of RASSF1A and DOK7 mRNA significantly increased (P<0.05). Our findings reveal for the first time the impact of DSCR1 ectopic expression on the methylation status of breast cancer cells and identify a novel agent for epigenetic therapy.     

DNA methylation and breast tumor clinicopathological features: The Western New York Exposures and Breast Cancer (WEB) study

We evaluated the association between methylation of 9 genes, SCGB3A1, GSTP1, RARB, SYK, FHIT, CDKN2A, CCND2, BRCA1, and SFN in tumor samples from 720 breast cancer cases with clinicopathological features of the tumors and survival. Logistic regression was used to estimate odds ratios (OR) of methylation and Cox proportional hazards models to estimate hazard ratios (HR) between methylation and breast cancer related mortality. Estrogen receptor (ER) and progesterone receptor (PR) positivity were associated with increased SCGB3A1 methylation among pre- and post-menopausal cases. Among premenopausal women, compared with Stage 0 cases, cases of invasive cancer were more likely to have increased methylation of RARB (Stage I OR = 4.7, 95% CI: 1.1–19.0; Stage IIA/IIB OR = 9.7, 95% CI: 2.4–39.9; Stage III/IV OR = 5.6, 95% CI: 1.1–29.4) and lower methylation of FHIT (Stage I OR = 0.2, 95% CI: 0.1–0.9; Stage IIA/IIB OR = 0.2, 95% CI: 0.1–0.8; Stage III/IV OR = 0.6, 95% CI: 0.1–3.4). Among postmenopausal women, methylation of SYK was associated with increased tumor size (OR = 1.7, 95% CI: 1.0–2.7) and higher nuclear grade (OR = 2.0, 95% CI 1.2–3.6). Associations between methylation and breast cancer related mortality were observed among pre- but not post-menopausal women. Methylation of SCGB3A1 was associated with reduced risk of death from breast cancer (HR = 0.41, 95% CI: 0.17–0.99) as was BRCA1 (HR = 0.41, 95% CI: 0.16–0.97). CCND2 methylation was associated with increased risk of breast cancer mortality (HR = 3.4, 95% CI: 1.1–10.5). We observed differences in methylation associated with tumor characteristics; methylation of these genes was also associated with breast cancer survival among premenopausal cases. Understanding of the associations of DNA methylation with other clinicopathological features may have implications for prevention and treatment.     

Different methylation and MicroRNA expression pattern in male and female familial breast cancer

Epigenetic regulation, has been very scarcely explored in familial breast cancer (BC). In the present study RASSF1A and RAR beta promoter methylation and miR17, miR21, miR 124, and let‐7a expression were investigated to highlight possible differences of epigenetic regulation between male and female familial BC, also in comparison with sporadic BC. These epigenetic alterations were studied in 56 familial BC patients (27 males and 29 females) and in 16 female sporadic cases. RASSF1A resulted more frequently methylated in men than women (76% vs. 28%, respectively, P = 0.0001), while miR17 and let‐7a expression frequency was higher in women than in men (miR17: 66% in women vs. 41% in men, P < 0.05; let‐7a: 45% in women vs. 15% in men, P = 0.015). RASSF1A methylation affected 27.6% of familial BC while 83% of familial cases showed high expression of the gene (P = 0.025); on the contrary, only 17% of familial BC presented RAR beta methylation and 55% of familial cases overexpressed this gene (P = 0.005). Moreover, miR17, miR21, and let‐7a resulted significantly overexpressed in familial compared to sporadic BC. RASSF1A overexpression (86% vs. 65%, P = 0.13) and RAR beta overexpression (57% vs. 32%, P = 0.11) were higher in BRCA1/2 carriers even if not statistical significance was reached. BRCA mutation carriers also demonstrated significant overexpression of: miR17 (93% vs. 35%, P = 0.0001), let‐7a (64% vs. 16%, P = 0.002), and of miR21 (100% vs. 65%, P = 0.008). In conclusion, the present data suggest the involvement of RASSF1A in familial male BC, while miR17 and let‐7a seem to be implied in familial female BC. J. Cell. Physiol. 228: 1264–1269, 2013. © 2012 Wiley Periodicals, Inc.     

Prognostic potential of <Emphasis Type="Italic">KLOTHO</Emphasis> and <Emphasis Type="Italic">SFRP1</Emphasis> promoter methylation in head and neck squamous cell carcinoma

Hypermethylation in the CpG island promoter regions of tumor suppressors is known to play a significant role in the development of HNSCC and the detection of which can aid the classification and prognosis of HNSCC. This study aims to profile the methylation patterns in a panel of key genes including CDKN2A, CDKN2B, KLOTHO (KL), RASSF1A, RARB, SLIT2, and SFRP1, in a group of HNSCC samples from Saudi Arabia. The extent of methylation in these genes is determined using the MethyLight assay and correlated with known clinicopathological parameters in our samples of 156 formalin-fixed and paraffin-embedded HNSCC tissues. SLIT2 methylation had the highest frequency (64.6%), followed by RASSF1A (41.3%), RARB (40.7%), SFRP1 (34.9), KL (30.7%), CKDN2B (29.6%), and CKDN2A (29.1%). KL and SFRP1 methylation were more predominant in nasopharyngeal tumors (P = 0.001 and P = 0.031 respectively). Kaplan Meier analysis showed that patients with moderately differentiated tumors who display SFRP1 methylation have significantly worse overall survival in comparison with other samples. In contrast, better clinical outcomes were seen in patients with KL methylation. In conclusion, our findings suggest that the detection of frequent methylation in SFRP1 and KL genes’ promoters could serve as prognostic biomarkers for HNSCC.     

RASSF2 methylation is a strong prognostic marker in younger age patients with Ewing sarcoma

Ras-association domain family of genes consist of 10 members (RASSF1-RASSF10), all containing a Ras-association (RA) domain in either the C- or the N-terminus. Several members of this gene family are frequently methylated in common sporadic cancers; however, the role of the RASSF gene family in rare types of cancers, such as bone cancer, has remained largely uninvestigated. In this report, we investigated the methylation status of RASSF1A and RASSF2 in Ewing sarcoma (ES). Quantitative real-time methylation analysis (MethyLight) demonstrated that both genes were frequently methylated in Ewing sarcoma tumors (52.5% and 42.5%, respectively) as well as in ES cell lines and gene expression was upregulated in methylated cell lines after treatment with 5-aza-2′-deoxcytidine. Overexpression of either RASSF1A or RASSF2 reduced colony formation ability of ES cells. RASSF2 methylation correlated with poor overall survival (p = 0.028) and this association was more pronounced in patients under the age of 18 y (p = 0.002). These results suggest that both RASSF1A and RASSF2 are novel epigenetically inactivated tumor suppressor genes in Ewing sarcoma and RASSF2 methylation may have prognostic implications for ES patients.     

Methylation-mediated regulation of the glutathione S-transferase P1 gene in human breast cancer cells

Understanding the mechanisms that regulate the human pi class GST (GSTP1) gene expression in breast cancer cells is of particular importance to the study of breast cancer biology. In cultured human breast cancer cell lines, GSTP1 is exclusively expressed in estrogen receptor-negative (ER−) cells but is undetectable in receptor-positive (ER+) cells. Previously, we examined transiently transfected GSTP1 promoter activities, in vitro GSTP1 promoter–DNA interactions, and GSTP1 mRNA stability. These studies indicated that transiently transfected GSTP1 promoter elements and GSTP1 mRNA stability could only partially explain cell line-specific expression of endogenous GSTP1. In the present study, we examined whether the methylation status of the GSTP1 CpG island plays an important role in GSTP1 regulation. Southern blot analysis revealed that the GSTP1 CpG island is hypermethlyated in ER+, GSTP1 non-expressing cell lines but is undermethylated in ER−, GSTP1 expressing cell lines. Moreover, partial demethylation of the GSTP1 CpG island by treatment with 5-aza-2′-deoxycytidine resulted in de novo gene expression in ER+ cell lines, as detected by RT-PCR, Northern blot and Western blot analyses. Our data strongly indicate that methylation status of the promoter contributes significantly to the levels of GSTP1 expressed in ER− and ER+ breast cancer cell lines.     

RASSF6 exhibits promoter hypermethylation in metastatic melanoma and inhibits invasion in melanoma cells

Brain metastasis is a major contributor to cancer mortality, yet, the genetic changes underlying the development of this capacity remain poorly understood. RASSF proteins are a family of tumor suppressors that often suffer epigenetic inactivation during tumorigenesis. However, their epigenetic status in brain metastases has not been well characterized. We have examined the promoter methylation of the classical RASSF members (RASSF1A-RASSF6) in a panel of metastatic brain tumor samples. RASSF1A and RASSF2 have been shown to undergo promoter methylation at high frequency in primary lung and breast tumors and in brain metastases. Other members exhibited little or no methylation in these tumors. In examining melanoma metastases, however, we found that RASSF6 exhibits the highest frequency of inactivation in melanoma and in melanoma brain metastases. Most melanomas are driven by an activating mutation in B-Raf. Introduction of RASSF6 into a B-Raf^V600E-containing metastatic melanoma cell line inhibited its ability to invade through collagen and suppressed MAPK pathway activation and AKT. RASSF6 also appears to increase the association of mutant B-Raf and MST1, providing a potential mechanism by which RASSF6 is able to suppress MAPK activation. Thus, we have identified a novel potential role for RASSF6 in melanoma development. Promoter methylation leading to reduced expression of RASSF6 may play an important role in melanoma development and may contribute to brain metastases.