Induction of Apoptosis and Autophagy Using Ectopic DSCR1 Expression in Breast Cancer Cells

Down syndrome critical region 1 gene (DSCR1) is an anti-angiogenesis gene that inhibits the growth of tumor cells. In this study, the role of autophagy and apoptosis in DSCR1-induced cytotoxicity were investigated in MDA-MB-468 breast cancer cells. Lentivirus vector harboring DSCR1 (LV-DSCR1⁺) was constructed in HEK 293 cells and the optimal dosage of lentivirus vector for infection was determined by the MTT assay. After infection of cells using LV-DSCR1⁺, acridine orange and ethidium bromide staining was performed to investigation of apoptosis and autophagy. Expression of DSCR1 and marker genes for angiogenesis (VEGF), apoptosis (Bax and Bcl2) and autophagy (LC3 and Beclin) were determined by Real time PCR. The cellular morphological changes related to apoptosis and autophagy was happened after 48 hours of viral infection. Fragmented bright orange nucleuses and vacuoles were observed due to the cell apoptosis and autophagy after acridine orange and ethidium bromide staining. Upregulation of Bax, Lc3, DSCR1 and Beclin1 and downregulation of Bcl2 and VEGF was detected due to treatment with LV-DSCR1⁺. These results demonstrated that LV-DSCR1⁺ can induce apoptosis and autophagy, therefore suggesting that it may serves as an efficient tool to breast cancer treatment.     

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.     

Integrin alpha9 (ITGA9) expression and epigenetic silencing in human breast tumors

Integrin alpha9 (ITGA9) is one of the less studied integrin subunits that facilitates accelerated cell migration and regulates diverse biological functions such as angiogenesis, lymphangiogenesis, cancer cell proliferation and migration. In this work, integrin alpha9 expression and its epigenetic regulation in normal human breast tissue, primary breast tumors and breast cancer cell line MCF7 were studied. It was shown that integrin alpha9 is expressed in normal human breast tissue. In breast cancer, ITGA9 expression was downregulated or lost in 44% of tumors while another 45% of tumors showed normal or increased ITGA9 expression level (possible aberrations in the ITGA9 mRNA structure were supposed in 11% of tumors). Methylation of ITGA9 CpG-island located in the first intron of the gene was shown in 90% of the breast tumors with the decreased ITGA9 expression while no methylation at 5′-untranslated region of ITGA9 was observed. 5-aza-dC treatment restored integrin alpha9 expression in ITGA9-negative MCF7 breast carcinoma cells, Trichostatin A treatment did not influenced it but a combined treatment of the cells with 5-aza-dC/Trichostatin A doubled the ITGA9 activation. The obtained results suggest CpG methylation as a major mechanism of integrin alpha9 inactivation in breast cancer with a possible involvement of other yet unidentified molecular pathways.Key words: integrin alpha9, ITGA9, breast cancer, expression, methylation, bisulfite sequencing     

Epigenetic changes of CXCR4 and its ligand CXCL12 as prognostic factors for sporadic breast cancer

Chemokines and their receptors are involved in the development and cancer progression. The chemokine CXCL12 interacts with its receptor, CXCR4, to promote cellular adhesion, survival, proliferation and migration. The CXCR4 gene is upregulated in several types of cancers, including skin, lung, pancreas, brain and breast tumors. In pancreatic cancer and melanoma, CXCR4 expression is regulated by DNA methylation within its promoter region. In this study we examined the role of cytosine methylation in the regulation of CXCR4 expression in breast cancer cell lines and also correlated the methylation pattern with the clinicopathological aspects of sixty-nine primary breast tumors from a cohort of Brazilian women. RT-PCR showed that the PMC-42, MCF7 and MDA-MB-436 breast tumor cell lines expressed high levels of CXCR4. Conversely, the MDA-MB-435 cell line only expressed CXCR4 after treatment with 5-Aza-CdR, which suggests that CXCR4 expression is regulated by DNA methylation. To confirm this hypothesis, a 184 bp fragment of the CXCR4 gene promoter region was cloned after sodium bisulfite DNA treatment. Sequencing data showed that cell lines that expressed CXCR4 had only 15% of methylated CpG dinucleotides, while the cell line that not have CXCR4 expression, had a high density of methylation (91%). Loss of DNA methylation in the CXCR4 promoter was detected in 67% of the breast cancer analyzed. The absence of CXCR4 methylation was associated with the tumor stage, size, histological grade, lymph node status, ESR1 methylation and CXCL12 methylation, metastasis and patient death. Kaplan-Meier curves demonstrated that patients with an unmethylated CXCR4 promoter had a poorer overall survival and disease-free survival. Furthermore, patients with both CXCL12 methylation and unmethylated CXCR4 had a shorter overall survival and disease-free survival. These findings suggest that the DNA methylation status of both CXCR4 and CXCL12 genes could be used as a biomarker for prognosis in breast cancer.     

<Emphasis Type="Italic">RASSF1A</Emphasis> expression level in primary epithelial tumors of various locations

Tumor suppressor activity of RASSF1A in vitro and in vivo was established, in particular, in studies of knockout mice cells. Data on methylation of the promoter region and a lower expression of RASSF1A were mostly obtained with cancer cell lines. Here, the RASSF1A mRNA was quantified the first time in primary epithelial malignant tumors of five various locations from 130 patients by semi-quantitative RT-PCR. Representative samples of kidney, lung, and breast carcinomas were examined. Preliminary data were obtained for RASSF1A expression in ovarian and colorectal carcinomas. System studies showed unexpected expression profiles, namely, the mRNA level increased (two- to sevenfold) more frequently than decreased in renal, breast, ovarian, and colorectal carcinomas. A higher RASSF1A mRNA level was significantly more frequent in renal cell carcinomas (24/38, 63% vs 8/38, 21%, P = 0.0004 by Fisher’s exact test) and ovarian carcinomas (8/13, 62% vs 2/13, 15%, P = 0.0114). Equal frequencies of lower and higher RASSF1A expression levels were only observed in non-small cell lung cancer (16/38, 42%). Noteworthy, an increase in expression was more common at early clinical stages of squamous cell lung cancer and adenocarcinoma, while a decrease in RASSF1A expression was more frequent at advanced clinical stages. In clear cell renal cell carcinoma, an increase in RASSF1A expression occurred more often at both early and advanced stages and was significant at advanced stages (P = 0.0094). The findings suggested tumor specificity for changes in RASSF1A expression. The observed regularities may also indicate that RASSF1A has dual functions in tumors, acting as a tumor suppressor and as a protooncogene.     

Increased promoter methylation in exfoliated breast epithelial cells in women with a previous breast biopsy

Accurately identifying women at increased risk of developing breast cancer will provide greater opportunity for early detection and prevention. DNA promoter methylation is a promising biomarker for assessing breast cancer risk. Breast milk contains large numbers of exfoliated epithelial cells that are ideal for methylation analyses. Exfoliated epithelial cells were isolated from the milk obtained from each breast of 134 women with a history of a non-proliferative benign breast biopsy (Biopsy Group). Promoter methylation of three tumor suppressor genes, RASSF1, SFRP1 and GSTP1, was assessed by pyrosequencing of bisulfite-modified DNA. Methylation scores from the milk of the 134 women in the Biopsy Group were compared to scores from 102 women for whom a breast biopsy was not a recruitment requirement (Reference Group). Mean methylation scores for RASSF1 and GSTP1 were significantly higher in the Biopsy than in the Reference Group. For all three genes the percentage of outlier scores was greater in the Biopsy than in the Reference Group but reached statistical significance only for GSTP1. A comparison between the biopsied and non-biopsied breasts of the Biopsy Group revealed higher mean methylation and a greater number of outlier scores in the biopsied breast for both SFRP1 and RASSF1, but not for GSTP1. This is the first evidence of CpG island methylation in tumor suppressor genes of women who may be at increased risk of developing breast cancer based on having had a prior breast biopsy.     

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.     

RASSF1A and DOK1 Promoter Methylation Levels in Hepatocellular Carcinoma,Cirrhotic and Non-Cirrhotic Liver,and Correlation with Liver Cancer in Brazilian Patients

Hepatocellular carcinoma (HCC) is the second most common cause of cancer mortality worldwide. Most cases of HCC are associated with cirrhosis related to chronic hepatitis B virus or hepatitis C virus infections. Hypermethylation of promoter regions is the main epigenetic mechanism of gene silencing and has been involved in HCC development. The aim of this study was to determine whether aberrant methylation of RASSF1A and DOK1 gene promoters is associated with the progression of liver disease in Brazilian patients. Methylation levels were measured by pyrosequencing in 41 (20 HCC, 9 cirrhotic, and 12 non-cirrhotic) liver tissue samples. Mean rates of methylation in RASSF1A and DOK1 were 16.2% and 12.0% in non-cirrhotic, 26.1% and 19.6% in cirrhotic, and 59.1% and 56.0% in HCC tissues, respectively, showing a gradual increase according to the progression of the disease, with significantly higher levels in tumor tissues. In addition, hypermethylation of RASSF1A and DOK1 was found in the vast majority (88%) of the HCC cases. Interestingly, DOK1 methylation levels in HCC samples were significantly higher in the group of younger (<40 years) patients, and higher in moderately differentiated than in poorly differentiated tumors (p < 0.05). Our results reinforce the hypothesis that hypermethylation of RASSF1A and DOK1 contributes to hepatocarcinogenesis and is associated to clinicopathological characteristics. RASSF1A and DOK1 promoter hypermethylation may be a valuable biomarker for early diagnosis of HCC and a potential molecular target for epigenetic-based therapy.     

Epigenetic regulation of DACH1, a novel Wnt signaling component in colorectal cancer

Colorectal cancer (CRC) is one of the common malignant tumors worldwide. Both genetic and epigenetic changes are regarded as important factors of colorectal carcinogenesis. Loss of DACH1 expression was found in breast, prostate, and endometrial cancer. To analyze the regulation and function of DACH1 in CRC, 5 colorectal cancer cell lines, 8 cases of normal mucosa, 15 cases of polyps and 100 cases of primary CRC were employed in this study. In CRC cell lines, loss of DACH1 expression was correlated with promoter region hypermethylation, and re-expression of DACH1 was induced by 5-Aza-2'-deoxyazacytidine treatment. We found that DACH1 was frequently methylated in primary CRC and this methylation was associated with reduction in DACH1 expression. These results suggest that DACH1 expression is regulated by promoter region hypermethylation in CRC. DACH1 methylation was associated with late tumor stage, poor differentiation, and lymph node metastasis. Re-expression of DACH1 reduced TCF/LEF luciferase reporter activity and inhibited the expression of Wnt signaling downstream targets (c-Myc and cyclinD1). In xenografts of HCT116 cells in which DACH1 was re-expressed, tumor size was smaller than in controls. In addition, restoration of DACH1 expression induced G2/M phase arrest and sensitized HCT116 cells to docetaxel. DACH1 suppresses CRC growth by inhibiting Wnt signaling both in vitro and in vivo. Silencing of DACH1 expression caused resistance of CRC cells to docetaxel. In conclusion, DACH1 is frequently methylated in human CRC and methylation of DACH1 may serve as detective and prognostic marker in CRC.     

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.     

Decreased expression of EZH2 reactivates RASSF2A by reversal of promoter methylation in breast cancer cells

EZH2, the catalytic subunit of polycomb repressor complex 2, has oncogenic properties, whereas RASSF2A, a Ras association domain family protein, has a tumor suppressor role in many types of human cancer. However, the interrelationship between these two genes remains unclear. Here, we showed that the downregulation of EZH2 reduces CpG island methylation of the RASSF2A promoter, thereby leading to increased RASSF2A expression. Our findings also showed that knockdown of EZH2 increased RASSF2A expression in the human breast cancer cell line MCF‐7 in cooperation with DNMT1. This was similar to the effect of 5‐Aza‐CdR, a DNA methylation inhibitor that reactivates tumor suppressor genes and activated RASSF2A expression in our study. The EZH2 inhibitor DZNep markedly suppressed the proliferation, migration, and invasion of MCF‐7 cells treated with ADR and TAM. EZH2 inhibits the expression of tumor suppressor gene RASSF2A via promoter hypermethylation. Thus, it plays an important role in tumorigenesis and is a potential therapeutic target for the treatment of breast cancer.     

Impact of Hyperhomocysteinemia on Breast Cancer Initiation and Progression: Epigenetic Perspective

Our recent study showing association of hyperhomocysteinemia and hypomethioninemia in breast cancer and other studies indicating association of hyperhomocysteinemia with metastasis and development of drug resistance in breast cancer cells treated with homocysteine lead us to hypothesize that homocysteine might modulate the expression of certain tumor suppressors, i.e., RASSF1, RARβ1, CNND1, BRCA1, and p21, and might influence prognostic markers such as BNIP3 by inducing epigenetic alteration. To demonstrate this hypothesis, we have treated MCF-7 and MDA-MB-231 cells with different doses of homocysteine and observed dose-dependent inhibition of BRCA1 and RASSF1, respectively. In breast cancer tissues, we observed the following expression pattern: BNIP3 > BRCA1 > RARβ1 > CCND1 > p21 > RASSF1. Hyperhomocysteinemia was positively associated with BRAC1 hypermethylation both in breast cancer tissue and corresponding peripheral blood. Peripheral blood CpG island methylation of BRCA1 in all types of breast cancer and methylation of RASSF1 in ER/PR-negative breast cancers showed positive correlation with total plasma homocysteine. The methylation of RASSF1 and BRCA1 was associated with breast cancer initiation as well as progression, while BRCA1 methylation was associated with DNA damage. Vitamin B₁₂ showed inverse association with the methylation at both the loci. RFC1 G80A and cSHMT C1420T variants showed positive association with methylation at both the loci. Genetic variants influencing remethylation step were associated positively with BRCA1 methylation and inversely with RASSF1 methylation. GCPII C1561T variant showed inverse association with BRCA1 methylation. We found good correlation of BRAC1 (r = 0.90) and RASSF1 (0.92) methylation pattern between the breast cancer tissue and the corresponding peripheral blood. To conclude, elevated homocysteine influences methionine dependency phenotype of breast cancer cells and is associated with breast cancer progression by epigenetic modulation of RASSF1 and BRCA1 .     

Culturing conditions highly affect DNA methylation and gene expression levels in MCF7 breast cancer cell line

The levels of DNA methylation and their role in gene expression are key factors that could affect diagnosis, prognosis, and treatment options of different diseases. In this study, the methylation levels of 22 genes that are mostly correlated to breast cancer were determined using EpiTect methyl II PCR array. This analysis was performed to determine the effect of cells’ passage number and the use of antibiotics in the culturing media on gene methylation levels in MCF7 cell line. DNA methylation levels of PTGS2, ADAM23, HIC1, and PYCARD were found to be significantly different among different passages. While the DNA methylation levels of CCNA1, RASSF1, and THBS1 were found to be affected by the use of 1% of penicillin/streptomycin in the culture media. Gene expression analysis after demethylation using 5-Aza-2′-deoxycytidine showed that the gene expression levels of the hypermethylated genes varied between different passage numbers. This study shows that the presence of antibiotic within cultured media and cell line’s passage number could greatly affect the methylation levels that need to be considered in future studies on cell lines.     

Modulation of protein expression levels and DNA methylation status of breast cancer metastasis genes by anthracycline-based chemotherapy and the demethylating agent decitabine

Epigenetic drugs are promising add‐ons to cancer treatment; still, adverse effects concerning tumour promotion have been reported occasionally. In this in vitro study, we investigated the effect of combination treatment of decitabine with anthracycline‐based chemotherapy [5‐fluorouracil plus epirubicine plus cyclophosphamide (FEC)] on viability and metastatic activity of breast cancer cell lines, MDA‐MB‐231 (estrogen receptor‐negative) and MCF‐7 (estrogen receptor‐positive). The effect of decitabine and its combined treatment with FEC on viability of both cancer cell lines was assessed using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazoliumbromide and adenosine triphosphate (ATP) cell survival assays. DNA methylation specific real‐time polymerase chain reaction (PCR) (Methylight®) was employed to document the methylation status of the metastasis‐relevant urokinase‐type plasminogen activator (uPA) and plasminogen activator inhibitor‐I (PAI‐1) genes. Additionally, protein expression levels of uPA and PAI‐1 were determined using enzyme‐linked immunosorbent assays. Invasion capacity of cells was assayed using Matrigel® invasion assay. Decitabine lowered the viability of MCF‐7 cells, although MDA‐MB‐231 cells were not affected. Decitabine did not augment FEC‐mediated cytotoxicity in both cell lines. In MCF‐7 cells, methylation of the uPA and PAI‐1 gene promoter was significantly reduced by decitabine or decitabine plus FEC. Protein levels of uPA and PAI‐1 were induced by all treatments. Decitabine significantly induced the invasion capacity of MCF‐7 cells, whereas all of the drugs resulted in decreased invasion capacity of MDA‐MB‐231. Our results suggest differential effects of single‐dose decitabine and its combination with FEC on the metastatic capacity and survival of breast cancer cell lines endowed with different metastatic behaviour. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

Development of a multiplex methylation specific PCR suitable for (early) detection of non-small cell lung cancer

Lung cancer is a worldwide health problem and a leading cause of cancer-related deaths. Silencing of potential tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event in the initiation and development of cancer. Thus, methylated cancer type-specific TSGs in DNA can serve as useful biomarkers for early cancer detection. We have now developed a “Multiplex Methylation Specific PCR” (MMSP) assay for analysis of the methylation status of multiple potential TSGs by a single PCR reaction. This method will be useful for early diagnosis and treatment outcome studies of non-small cell lung cancer (NSCLC). Genome-wide CpG methylation and expression microarrays were performed on lung cancer tissues and matched distant non-cancerous tissues from three NSCLC patients from China. Thirty-eight potential TSGs were selected and analyzed by methylation PCR on bisulfite treated DNA. On the basis of sensitivity and specificity, six marker genes, HOXA9, TBX5, PITX2, CALCA, RASSF1A, and DLEC1, were selected to establish the MMSP assay. This assay was then used to analyze lung cancer tissues and matched distant non-cancerous tissues from 70 patients with NSCLC, as well as 24 patients with benign pulmonary lesion as controls. The sensitivity of the assay was 99% (69/70). HOXA9 and TBX5 were the 2 most sensitive marker genes: 87% (61/70) and 84% (59/70), respectively. RASSF1A and DLEC1 showed the highest specificity at 99% (69/70). Using the criterion of identifying at least any two methylated marker genes, 61/70 cancer samples were positive, corresponding to a sensitivity of 87% and a specificity of 94%. Early stage I or II NSCLC could even be detected with a 100% specificity and 86% sensitivity. In conclusion, MMSP has the potential to be developed into a population-based screening tool and can be useful for early diagnosis of NSCLC. It might also be suitable for monitoring treatment outcome and recurrence.