Tumor Suppressor Gene RASSF1A Research Articles

Polymorphism of A133S and promoter hypermethylation in Ras association domain family 1A gene (RASSF1A) is associated with risk of esophageal and gastric cardia cancers in Chinese population from high incidence area in northern China

BackgroundThe role of tumor suppressor gene RASSF1A in the esophageal and gastric cardia carcinogenesis is still inconclusive. In this study, the polymorphism, promoter methylation and gene expression of RASSF1A were characterized in esophageal squamous cell carcinoma (ESCC) and gastric cardia adenocarcinoma (GCA).MethodsWe firstly analyzed the prevalence of RASSF1A A133S in a total of 228 cancer patients with ESCC (n=112) and GCA (n=116) and 235 normal controls by polymerase chain reaction (PCR) and restriction enzyme-digestion assay. Then, the promoter methylation status of the RASSF1A in ESCC (n=143), GCA (n=92) and corresponding adjacent normal tissues were further investigated using methylation-specific PCR (MSP) approach. Finally, the RASSF1A protein expression were determined in ESCC (n=27), GCA (n=24) and the matched adjacent normal tissues by immunohistochemical method.ResultsThe frequency of 133Ala/Se and Ser/Ser genotype was significantly higher in GCA patients than in normal controls (19.0% vs. 10.2%, P=0.02). Compared with Ala/Ala genotype, Ala/Se and Ser/Ser genotype significantly increased susceptibility to GCA (OR=2.06, 95% CI=1.09–3.97). However, this polymorphism had no association with ESCC (P=0.69). The promoter methylation of RASSF1A gene was significantly increased the risk to both ESCC (OR=5.90, 95% CI=2.78–12.52) and GCA (OR=7.50, 95% CI= 2.78–20.23). Promoter methylation of RASSF1A gene in ESCC was also associated with age and cancer cell differentiation (for age: OR=3.11, 95% CI=1.10–8.73; for differentiation: OR=0.29, 95% CI=0.12–0.69). RASSF1A positive expression was significantly decreased the risk of GCA (OR=0.16, 95% CI=0.03–0.83). In contrast, there was no statistical significance between RASSF1A positive expression and ESCC. The expression of RASSF1A protein trend to be positively related with older GCA patients (OR=16.20, 95% CI=1.57–167.74).ConclusionsThe present findings suggest that alterations of RASSF1A may play an important role in gastric cardia carcinogenesis in terms of polymorphism, promoter hypermethylation and protein expression. Whereas, RASSF1A hypermethylation may probably also be involved in esophageal squamous cell carcinogenesis.

Abstract 656: Promoter hypermethylation and silencing of tumor suppressor genes RASSF1A, WIF1, SLIT2 and ROBO1 in triple negative breast carcinomas.

Abstract Triple negative breast cancers represent 10-17% of all breast carcinomas. They display a high histological grade, a more aggressive clinical behavior and they lack any specific treatment, thus are associated to a bad prognosis. As other cancer types, this cancer is caused by sequential accumulation of mutations and genomic alterations involving tumor suppressor genes and oncogenes. In this matter, inactivation of tumor suppressor genes may occur by deletion, promoter hypermethylation or point mutations. In previous study by array CGH we found several tumor suppressor candidate genes deleted in a hereditary breast cancer set of tumors. In addition we studied 4 of these tumor suppressor genes RASSF1A, WIF1, SLIT2 and ROBO1 in a non triple negative set of breast cancer tumors, and found a high frequency of promoter methylation, statistically associated with loss of protein expression for RASSF1A and WIF1. In the present study we aimed to extend the analysis to triple negative breast cancer tumors due to their clinical relevance. Methylation analysis through MS-PCR revealed that RASSF1A promoter was hypermethylated in 52.9% of triple negative tumors, and presents loss of expression in 80.6% of the tumors. WIF1 promoter was hypermethylated in 89.1% and its protein expression was downregulated in 63.1% of the tumors. For SLIT2 we found frequent promoter hypermethylation among tumors (82.6%) showing loss of expression in 88% of the tumors. MS-PCR analyses were not informative for ROBO1, but protein expression was reduced in 49% of all tumors. Our results show that a high percentage of tumors present silencing of RASSF1A, WIF1, SLIT2 and/or ROBO1, and therefore implying the impairment of the pathways they represent. This findings support these genes may have an important role in triple negative breast tumor development. Fondecyt 1080595. Citation Format: Carolina Alvarez, Gloria Garrido, Patricia Gajardo, Alonso Quiroz, Valeria Cornejo, Wanda Fernandez, Mauricio Camus, Pilar Carvallo. Promoter hypermethylation and silencing of tumor suppressor genes RASSF1A, WIF1, SLIT2 and ROBO1 in triple negative breast carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 656. doi:10.1158/1538-7445.AM2013-656

Promoter hypermethylation of the tumor-suppressor genes ITIH5, DKK3, and RASSF1A as novel biomarkers for blood-based breast cancer screening

IntroductionFor early detection of breast cancer, the development of robust blood-based biomarkers that accurately reflect the host tumor is mandatory. We investigated DNA methylation in circulating free DNA (cfDNA) from blood of breast cancer patients and matched controls to establish a biomarker panel potentially useful for early detection of breast cancer.MethodsWe examined promoter methylation of seven putative tumor-suppressor genes (SFRP1, SFRP2, SFRP5, ITIH5, WIF1, DKK3, and RASSF1A) in cfDNA extracted from serum. Clinical performance was first determined in a test set (n = 261 sera). In an independent validation set (n = 343 sera), we validated the most promising genes for further use in early breast cancer detection. Sera from 59 benign breast disease and 58 colon cancer patients were included for additional specificity testing.ResultsBased on the test set, we determined ITIH5 and DKK3 promoter methylation as candidate biomarkers with the best sensitivity and specificity. In both the test and validation set combined, ITIH5 and DKK3 methylation achieved 41% sensitivity with a specificity of 93% and 100% in healthy and benign disease controls, respectively. Combination of these genes with RASSF1A methylation increased the sensitivity to 67% with a specificity of 69% and 82% in healthy controls and benign disease controls, respectively.ConclusionsTumor-specific methylation of the three-gene panel (ITIH5, DKK3, and RASSF1A) might be a valuable biomarker for the early detection of breast cancer.

Abstract A36: Resveratrol decreases DNMTs in a human relevant animal model of breast cancer

Abstract Background: We previously reported that trans-resveratrol (resveratrol) downregulates the expression DNA methyltransferases (DNMTs) 1 and 3b in vitro and demethylates the tumor suppressor gene RASSF-1a in the breasts of women who are at increased breast cancer risk. Effects increased with increasing dose/circulating level of the agent, respectively. In the current report, we evaluated the effect of increasing doses of resveratrol on mammary tumor formation in the ACI rat, which develops estrogen dependent mammary carcinoma. Methods: 91 female rats were treated with a slow release estradiol (E2) pellet plus one of the following: 5 mg/kg/day (low dose) resveratrol (n=23), 25 mg/kg/day (high dose) resveratrol (n=22), 0.1mg/kg/week 5-azacytadine (aza) (n=22), an inhibitor of DNMTs which is used clinically, or control (n=24). The animals were examined weekly to assess for the presence of tumor formation. After 5.5 months of treatment, the animals were sacrificed and the mammary glands harvested, with half of each gland snap frozen and the other half formalin fixed. Specimens were evaluated by a board certified pathologist to confirm histology as benign or malignant. DNMT1 and 3b were measured by qRT-PCR. Results: Tumor formation was lowest (18% of rats) in the high dose resveratrol and 5-aza groups, intermediate in the low dose resveratrol group (26%) and highest in control (33%). DMNT1 expression was significantly (p<0.0001) influenced by treatment, while DNMT3b was not. On the other hand, DNMT3b expression was significantly (p<0.0001) influenced by histology (benign vs. malignant), whereas DNMT1 was not. Conclusion: Treatment with resveratrol and 5-aza significantly influence DNMT expression, which may be important in the prevention/delay of mammary tumor formation. This mechanism of action of resveratrol warrants further investigation. Citation Format: Wenyi Qin, Weizhu Zhu, Ke Zhang, Kaitlin Clarke, Edward R. Sauter. Resveratrol decreases DNMTs in a human relevant animal model of breast cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A36.

Loss of RASSF1A synergizes with deregulated RUNX2 signaling in tumorigenesis.

The tumor suppressor gene RASSF1A is inactivated through point mutation or promoter hypermethylation in many human cancers. In this study, we conducted a Sleeping Beauty transposon-mediated insertional mutagenesis screen in Rassf1a-null mice to identify candidate genes that collaborate with loss of Rassf1a in tumorigenesis. We identified 10 genes, including the transcription factor Runx2, a transcriptional partner of Yes-associated protein (YAP1) that displays tumor suppressive activity through competing with the oncogenic TEA domain family of transcription factors (TEAD) for YAP1 association. While loss of RASSF1A promoted the formation of oncogenic YAP1-TEAD complexes, the combined loss of both RASSF1A and RUNX2 further increased YAP1-TEAD levels, showing that loss of RASSF1A, together with RUNX2, is consistent with the multistep model of tumorigenesis. Clinically, RUNX2 expression was frequently downregulated in various cancers, and reduced RUNX2 expression was associated with poor survival in patients with diffuse large B-cell or atypical Burkitt/Burkitt-like lymphomas. Interestingly, decreased expression levels of RASSF1 and RUNX2 were observed in both precursor T-cell acute lymphoblastic leukemia and colorectal cancer, further supporting the hypothesis that dual regulation of YAP1-TEAD promotes oncogenic activity. Together, our findings provide evidence that loss of RASSF1A expression switches YAP1 from a tumor suppressor to an oncogene through regulating its association with transcription factors, thereby suggesting a novel mechanism for RASSF1A-mediated tumor suppression.

MiR-7 and miR-218 epigenetically control tumor suppressor genes RASSF1A and Claudin-6 by targeting HoxB3 in breast cancer

Many microRNAs have been implicated as key regulators of cellular growth and differentiation and have been found to dysregulate proliferation in human tumors, including breast cancer. Cancer-linked microRNAs also alter the epigenetic landscape by way of DNA methylation and post-translational modifications of histones. Aberrations in Hox gene expression are important for oncogene or tumor suppressor during abnormal development and malignancy. Although recent studies suggest that HoxB3 is critical in breast cancer, the putative role(s) of microRNAs impinging on HoxB3 is not yet fully understood. In this study, we found that the expression levels of miR-7 and miR-218 were strongly and reversely associated with HoxB3 expression. Stable overexpression of miR-7 and miR-218 was accompanied by reactivation of tumor suppressor genes including RASSF1A and Claudin-6 by means of epigenetic switches in DNA methylation and histone modification, giving rise to inhibition of the cell cycle and clone formation of breast cancer cells. The current study provides a novel link between overexpression of collinear Hox genes and multiple microRNAs in human breast malignancy.

Anti-Her2 single-chain antibody mediated DNMTs-siRNA delivery for targeted breast cancer therapy

The targeted delivery of small interfering RNA (siRNA) to specific tumor tissues and tumor cells remains as one of the key challenges in the development of RNA interference as a therapeutic application. To target breast cancer, we developed a therapeutic delivery system using a fusion protein of an anti-Her2 single-chain antibody fragment with a positively charged protamine, namely F5-P, as the carrier to specifically deliver siRNA-targeting DNA methyltransferases 1 and/or 3b genes (siDNMTs) into Her2-expressing breast tumor cells. The carrier F5-P, expressed by the Escherichia coli system, was able to bind siRNA molecules and specifically deliver the siRNA to Her2-expressing BT474 breast cancer cells but not Her2-nonexpressing MDA-MB-231 breast cancer cells, while delivery of siDNMTs to BT474 cells successfully silenced the expression of targeted DNA methyltransferases (DNMTs) and facilitated the de-methylation of the RASSF1A tumor suppressor gene promoter, leading to the suppression of tumor cell proliferation. Moreover, as demonstrated in the BT474 xenograft murine model, F5-P successfully delivered siRNA into a Her2-expressing breast tumor, and tumor growth inhibition was mediated by an intravenous injection of F5-P/siDNMTs complex by down-regulating the expression of DNMTs and restoring tumor suppressor gene expression. These data suggest that the delivery of siDNMTs by F5-P could be used to treat Her2-expressing breast cancer.

Abstract 5424: Docosahexaenoic acid (DHA) induces gene expression of RASSF1A through epigenetics mechanisms in breast cancer cells

Abstract Background: Epigenetic changes, such as DNA methylation and post-translational histones modifications have an important role in mammary tumorigenesis. Changes in DNA methylation include global hypomethylation and hypermethylation of promoter regions of specific genes. Several genes are hypermethylated in breast cancer, especially the tumor suppressor gene RASSF1A, that have potential prognostic value. Epigenetic events are important as therapeutic targets due to its reversible feature. Dietary compounds can modulate the epigenetic processes in cancer. Experimentally, docosahexaenoic acid (DHA), a member of the omega-3 fatty acids, can reduce proliferation, induce apoptosis and decrease the invasive potential of breast tumor cells. However, the antitumor mechanisms of DHA and its ability to modulate epigenetic events are not yet fully elucidated. Objectives: To verify DHA action on RASSF1A expression and promoter region methylation in different human breast cancer cell lines. Material and Methods: Three different breast cell lines (MCF-7, MDA-MB-231, SKBR-3) were treated or not with 100 μM of DHA for 72 hours. Real time quantitative PCR were performed for gene expression quantification of RASSF1A. DNA methylation of the promoter region of RASSF1A was evaluated by methylation specific PCR. The western blot will be held for analyze of the histone 3 lysine 9 acetylated (H3K9ac) protein. Moreover, we will evaluate the phases of the cell cycle and apoptosis by flow cytometry. Results and Conclusion: Compared to control cells (not treated), DHA increased RASSF1A expression on MCF-7, but not in MDA-MB and in SKBR-3. These three transformed breast cells lines show methylation in specific region of RASSF1A promoter, indicating its role as a tumour supressing gene. DHA treatment increased RASSF1A promoter region hypomethylation in MCF-7, but not in MDA-MB and in SKBR-3. From this perspective, we can suggest that DHA could act by epigenetic mechanisms and reactivate RASSF1A, previously silenced. This action occured specifically in MCF-7, a less aggressive breast tumor cell line. It is expected that the present results promote better understanding of DHA role as a potential anti-oncogenic compound of breast 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 5424. doi:1538-7445.AM2012-5424

Abstract 4086: Location matters: The impact of the location of CpG sites on the specificity of methylation biomarkers for HCC

Abstract Hepatocellular carcinoma (HCC) is the second fastest growing cancer and one of the top 10 causes of cancer deaths in the United States. The 5-year survival rate is 14%, ranging from 26% among early stage tumors to only 2% in late stage HCC. The only available biomarker for screening, serum alpha-fetoprotein (AFP), has a low sensitivity of detection (40%-60%), so the need for a better detection method is urgent. Given the heterogeneous nature of HCC, a panel of genetic and epigenetic biomarkers would be needed to obtain high sensitivity for the early diagnosis of HCC. In the course of assembling a panel of epigenetic markers for HCC, we noticed variable HCC specificity of three extensively studied potential methylation biomarkers, APC, GSTP1, and RASSF1A. We hypothesized that the specificity of promoter methylation of the tumor suppressor genes APC, GSTP1, and RASSF1A as a biomarker for HCC could depend upon the location of the CpG sites analyzed. To test this hypothesis, we used bisulfite-PCR sequencing to compare the methylation status of the promoter region of these three genes in DNA isolated from HCC, matched adjacent non-HCC, cirrhosis, hepatitis, and normal liver. We identified HCC-specific CpG methylation sites and unique liver-specific methylation patterns in the promoter region of the APC (Jain et. al, PloSOne 2011) and GSTP1 genes and in a more HCC-specific promoter region (p < 0.05 by Fisher's exact test) of the RASSF1A gene. Together, these results proved our hypothesis and provided an interpretation for the variability in the literature. Using PCR-based technology, we have developed assays for a panel of genetic and epigenetic markers for liver cancer screening. These assays were first tested in tissue to determine their ability to distinguish HCC (n=120) from cirrhosis (n=35) and hepatitis (n=35). The AUROC is 0.809 for mAPC, 0.756 for mGSTP1, 0.887 for mRASSF1, and 0.733 for TP53. When combined, this panel of DNA markers had an AUROC of 0.931 and identified 90% of AFP-negative tumors. This study clearly demonstrates (1) the importance of location of CpG site methylation for HCC specificity and the way in which liver-specific DNA methylation should be considered when an epigenetic DNA marker is studied for detection of HCC and (2) the potential of a sensitive DNA test for HCC screening. An analysis of the performance of this panel of markers in peripherals (blood and urine) for HCC screening is in progress. 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 4086. doi:1538-7445.AM2012-4086

Abstract 581: Complementary reactivation of tumor suppressor genes in breast cancer cells by curcumin and curcumin O-glucuronide

Abstract Purpose: Curcumin appears to be a promising chemopreventive agent for various human cancers including breast cancer (BC). However, the molecular mechanism(s) by which it functions in vivo have not been fully elucidated. Recently, we have shown curcumin to be an effective hypomethylating agent in acute myeloid leukemia cells. Here, we investigated the ability of curcumin and its major metabolite, curcumin O-glucuronide (COG) to epigenetically modulate tumor suppressor gene expression in BC cells. Method: MCF-7 and MDA-MB-231 BC cells were treated with curcumin and COG (0.1 to 20 μM) for up to 96 hrs. MCF-7 and MDA-MB-231 cell engrafted tumor-bearing nude mice were treated with i.p. doses of 100 mg/kg curcumin or oral doses of 300 mg/kg nanoemulsion curcumin (NEC). The mRNA levels of RASSF1A, TIMP-3, DNMT1, microRNA29b, and microRNA221/222 were assessed by RT-PCR and protein levels were measured by Western Blot. DNA methylation levels were assessed using LC-MS. The in vitro anti-proliferative activities of curcumin, COG and NEC and the in vivo anti-tumor growth activities of curcumin and NEC were evaluated. Results: Curcumin induced significant global DNA hypomethylation in MCF-7 cells and reactivated RASSF1A expression in both MCF-7 and MDA-MB-231 cells. In vivo reactivation of RASSF1A by curcumin was confirmed in MCF-7 engrafted tumor tissues in mice. Curcumin reactivated TIMP-3 expression in MCF-7 engrafted tumor tissues, but down-regulated TIMP-3 expression in MCF-7 cells in vitro. We have evidence that this discrepancy was due to differential metabolism of curcumin to COG in vitro (no COG formation) and in vivo (COG level > curcumin), since COG itself reactivated TIMP-3 in MCF-7 cells. Notably, COG down-regulated microRNA221/222, two oncogenic microRNAs that suppress TIMP-3 expression, which may account for TIMP-3 reactivation in MCF-7 cells by COG. Importantly, COG chemically inhibited DNMT1 methylation activity and up-regulated microRNA29b, a central regulator of DNMTs in MCF-7 and MDA-MB-231 cells, which may be responsible for its hypomethylating activity. Curcumin elicited anti-tumor growth effects in both MCF-7 (70%) and MDA-MB-231 (27%) cell engrafted tumor bearing nude mice after four weeks of daily i.p. injection. Notably, NEC inhibited MDA-MB-231 cell engrafted tumor growth up to 50%, nearly twice that of curcumin. Conclusion: Curcumin reactivated RASSF1A and TIMP-3 tumor suppressor genes in vivo presumably through its conversion to COG and COG's modulation of DNA methylation and microRNAs. NEC is a more potent formulation for MDA-MB-231 cell engrafted tumor growth in nude mice when compared to curcumin. Further evaluation of NEC for BC prevention or treatment either alone or in combination with clinically-used agents is warranted. Supported by R21 CA135478 [ZL] and biomedical mass spectrometry laboratory (KKC and ZL). 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 581. doi:1538-7445.AM2012-581

RASSF1A and the Taxol Response in Ovarian Cancer

The RASSF1A tumor suppressor gene is frequently inactivated by promoter methylation in human tumors. The RASSF1A protein forms an endogenous complex with tubulin and promotes the stabilization of microtubules. Loss of RASSF1A expression sensitizes cells to microtubule destabilizing stimuli. We have observed a strong correlation between the loss of RASSF1A expression and the development of Taxol resistance in primary ovarian cancer samples. Thus, we sought to determine if RASSF1A levels could dictate the response to Taxol and whether an epigenetic therapy approach might be able to reverse the Taxol resistant phenotype of RASSF1A negative ovarian tumor cells. We found that knocking down RASSF1A expression in an ovarian cancer cell line inhibited Taxol-mediated apoptosis and promoted cell survival during Taxol treatment. Moreover, using a combination of small molecule inhibitors of DNA Methyl Transferase enzymes, we were able restore RASSF1A expression and Taxol sensitivity. This identifies a role for RASSF1A in modulating the tumor response to Taxol and provides proof of principal for the use of epigenetic therapy to overcome Taxol resistance.

Promoter methylation of MGMT, MLH1 and RASSF1A tumor suppressor genes in head and neck squamous cell carcinoma: Pharmacological genome demethylation reduces proliferation of head and neck squamous carcinoma cells

Promoter hypermethylation of tumor suppressor genes (TSGs) is a common feature of primary cancer cells. However, to date the somatic epigenetic events that occur in head and neck squamous cell carcinoma (HNSCC) tumorigenesis have not been well-defined. In the present study, we analyzed the promoter methylation status of the genes mutL homolog 1 (MLH1), Ras-association domain family member 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in 23 HNSCC samples, three control tissues and one HNSCC cell line (UM-SCC 33) using methylation-specific PCR (MSP). The expression of the three proteins was quantified by semi-quantitative immunohistochemical analysis. The cell line was treated with the demethylating agent 5-azacytidine (5-Aza) and the methylation status after 5-Aza treatment was analyzed by MSP and DNA sequencing. Proliferation was determined by Alamar blue staining. We found that the MGMT promoter in 57% of the analyzed primary tumor samples and in the cell line was hypermethylated. The MLH promoter was found to be methylated in one out of 23 (4%) tumor samples while in the examined cell line the MLH promoter was unmethylated. The RASSF1A promoter showed methylation in 13% of the tumor samples and in the cell line. MGMT expression in the group of tumor samples with a hypermethylated promoter was statistically significantly lower compared to the group of tumors with no measured hypermethylation of the MGMT promoter. After treatment of the cell line with the demethylating agent 5-Aza no demethylation of the methylated MGMT and RASSF1A genes were determined by MSP. DNA sequencing verified the MSP results, however, increased numbers of unmethylated CpG islands in the promoter region of MGMT and RASSF1A were observed. Proliferation was significantly (p<0.05) reduced after treatment with 5-Aza. In summary, we have shown promoter hypermethylation of the tumor suppressor genes MGMT and RASSF1A in HNSCC, suggesting that this epigenetic inactivation of TSGs may play a role in the development of HNSCC. 5-Aza application resulted in partial demethylation of the MGMT and RASSF1A TSGs and reduced proliferation of the tumor cells suggesting further evaluation of 5-Aza for HNSCC treatment.

Hepatitis C virus core upregulates the methylation status of the RASSF1A promoter through regulation of SMYD3 in hilar cholangiocarcinoma cells

Increasing evidence has been accumulated indicating the important role of epigenetic regulation in tumor genesis. Previously, we observed that the transfection of hepatitis C virus core (HCVc) protein led to malignant transformation in normal biliary cells, and that tumor suppressor gene RASSF1A was downregulated in many hilar cholangiocarcinoma patients by hypermethylation in the promoter region. In the present study, we found SET and MYND domain-containing protein 3 (SMYD3), a novel histone methyltransferase, was overexpressed in cholangiocarcinoma patients especially in those with HCV infection. Transfection of HCVc into hilar cholangiocarcinoma cell lines QBC939 and FRH0201 could upregulate the expression of SMYD3 and promote cell growth, which was consistent with the results of our clinical research. This phenomenon indicated that SMYD3 was related to the epigenetic regulation of cholangiocarcinoma genesis with HCV infection. Overexpression of SMYD3 could inhibit RASSF1A expression, whereas inhibition of SMYD3 by siRNA improved its expression. Methylation-specific polymerase chain reaction (MS-PCR) results showed the methylation status of RASSF1A promoter was regulated by SMYD3. In conclusion, HCVc could upregulate the methylation status of the RASSF1A promoter through regulation of SMYD3, and histone methylation may affect the DNA methylation of downstream gene by an unknown mechanism.

Methylation of tumor suppressor gene RASSF1A and BRCA1 in primary epithelial ovarian cancer

The methylation features of plasma tumor suppressor gene promoters in patients with primary Epithelial Ovarian Cancer (EOC) were analyzed in the present study. The methylation status of tumor suppressor genes BRCA1 and RASSF1A in primary EOC tissues and plasma was detected by Methylation-Specific polymerase chain reaction (MSP) to analyze the correlation of methylation and clinical as well as pathological parameters of EOC. The methylation proportion of tumor suppressor gene BRCA1 and RASSF1A was 17.1 and 42.9% in EOC tissues, respectively. There was positive correlation between BRCA1 methylation and histological stage, RASSF1A and clinical stage or histological stage. Meanwhile, the methylation proportion of BRCA1 and RASSF1A was 8.7 and 30.8% in the plasma of EOC patients, respectively. But, the methylation proportion of plasma BRCA1 and RASSF1A was 33.3 and 53.3% in EOC patients with abnormal BRCA1 and RASSF1A methylation in EOC tissues. The methylation ofBRCA1 and RASSF1A promoters may participate in the occurrence and development of primary EOC. The methylation proportion of plasma BRCA1 and RASSF1A is coincident with that in tumor tissues, and thus plasma DNA can effectively reflect the features of primary EOC. Key words: Ovarian neoplasms, plasma DNA, tumor suppressor gene, methylation, promoter.

RETRACTED: Aberrant DNA hypermethylation patterns lead to transcriptional silencing of tumor suppressor genes in UVB-exposed skin and UVB-induced skin tumors of mice

Overexposure of the human skin to solar ultraviolet (UV) radiation is the major etiologic factor for development of skin cancers. Here, we report the results of epigenetic modifications in UV-exposed skin and skin tumors in a systematic manner. The skin and tumor samples were collected after chronic exposure of the skin of SKH-1 hairless mice to UVB radiation using a well-established photocarcinogenesis protocol. We found a distinct DNA hypermethylation pattern in the UVB-exposed epidermal skin and UVB-induced skin tumors that was associated with the elevated expression and activity of the DNA methyltransferases (Dnmt) 1, Dnmt3a and Dnmt3b. To explore the role of hypermethylation in skin photocarcinogenesis, we focused on the p16(INK4a) and RASSF1A tumor suppressor genes, which are transcriptionally silenced on methylation. We established that the silencing of these genes in UVB-exposed epidermis and UVB-induced skin tumors is associated with a network of epigenetic modifications, including hypoacetylation of histone H3 and H4 and increased histone deacetylation, as well as recruitment of methyl-binding proteins, including MeCP2 and MBD1, to the methylated CpGs. Higher levels of DNA methylation and DNMT activity in human squamous cell carcinoma specimens than in normal human skin suggest that the data are relevant clinically. Our data indicate for the first time that UVB-induced DNA hypermethylation, enhanced Dnmt activity and histone modifications occur in UVB-exposed skin and UVB-induced skin tumors and suggest that these events are involved in the silencing of tumor suppressor genes and in skin tumor development.

The prognostic value of RASSF1A promoter hypermethylation in non-small cell lung carcinoma: a systematic review and meta-analysis

Inactivation of the tumor suppressor gene RASSF1A through methylation of the CpG islands within its promoter region as a prognostic factor for survival in non-small cell lung carcinoma (NSCLC) remains controversial. A meta-analysis of published studies investigating the effects of RASSF1A methylation on both relapse-free survival (RFS) and overall survival (OS) among NSCLC patients was performed. A total of 2802 patients from 19 eligible studies were included in the systematic review and 17 studies were included in the meta-analysis. In all, 32.6% of NSCLC patients had the methylated RASSF1A allele. Four of these studies investigated the correlation between RASSF1A methylation and RFS using univariate analysis. The univariate estimate for RFS was 1.87 [95% confidence interval (CI): 1.41-2.49; P < 0.0001] with no evidence of significant heterogeneity. Thirteen studies undertook univariate analyses of RASSF1A methylation and OS and 12 undertook multivariate analyses of RASSF1A methylation and OS. The pooled hazard ratio (HR) estimate for OS was 1.52 (95% CI: 1.33-1.74; P < 0.0001) by univariate analysis and 1.34 (95% CI: 1.15-1.57; P < 0.0001) by multivariate analysis. No significant heterogeneity was detected. For stages I-II NSCLC, the meta-risk remained highly significant by both univariate (HR = 1.94; 95% CI: 1.54-2.44; P < 0.0001) and multivariate analysis (HR = 1.39; 95% CI: 1.02-1.90; P = 0.039). This study shows that RASSF1A methylation appears to be an independent prognostic factor for poor survival in surgically treated NSCLC. However, the present findings require confirmation though adequately designed prospective studies.

Intratracheally Administered 5-Azacytidine Is Effective Against Orthotopic Human Lung Cancer Xenograft Models and Devoid of Important Systemic Toxicity

Introduction: Hypermethylation of key tumor suppressor genes plays an important role in lung carcinogenesis. The purpose of this study is to explore the therapeutic potential of regional administration (via the airways) of the demethylating agent 5-azacytidine (5-Aza) for the treatment of early lung cancer. Patients and MethodsWe administered 5-Aza solution directly into the trachea in imprinting control region (ICR) mice (to study its toxicity) and in nude mice bearing orthotopic human lung cancer xenografts (to assess its antitumor activity). ResultsIn vitro, 5-Aza inhibited the growth of human lung cancer cell lines H226, H358, and H460 in a dose-dependent manner. The concentrations to inhibit cell growth by 50% (IC50) were about 0.6-4.9 μg/mL. 5-Azacytidine reversed hypermethylation in the promoter of tumor suppressor gene RASSF1a in the H226 cells at a 6000-fold lower concentration than its IC50. In animal studies, intratracheal (I.T.) administration of 90 mg/kg 5-Aza (the maximum tolerated dose of 5-Aza intravenous injection [I.V.]) resulted in moderate pulmonary toxicity and 5-fold reduced myelosuppression compared with the same dose of I.V. 5-Aza. Using an optimized multiple dose schedule, I.T. 5-Aza was about 3-fold more effective than I.V. 5-Aza in prolonging the survival of mice bearing orthotopic H460 and H358 xenografts, and did not cause any detectable toxicity. Conclusion5-Azacytidine can reverse the hypermethylation in the human lung cancer cell lines at a nontoxic dose. Regional administration to the airways enhances the therapeutic index of 5-Aza by 75-fold. The potential of regional administration of 5-Aza (including by aerosolization) for the treatment of advanced bronchial premalignancy deserves further investigation.

Nanaomycin A Selectively Inhibits DNMT3B and Reactivates Silenced Tumor Suppressor Genes in Human Cancer Cells

Enzymes involved in the epigenetic regulation of the genome represent promising starting points for therapeutic intervention by small molecules, and DNA methyltransferases (DNMT) are emerging targets for the development of a new class of cancer therapeutics. In this work, we present nanaomycin A, initially identified by a virtual screening for inhibitors against DNMT1, as a compound inducing antiproliferative effects in three different tumor cell lines originating from different tissues. Nanaomycin A treatment reduced the global methylation levels in all three cell lines and reactivated transcription of the RASSF1A tumor suppressor gene. In biochemical assays, nanaomycin A revealed selectivity toward DNMT3B. To the best of our knowledge, this is the first DNMT3B-selective inhibitor identified to induce genomic demethylation. Our study thus establishes the possibility of selectively inhibiting individual DNMT enzymes.

CpG island hypermethylation of multiple tumor suppressor genes associated with loss of their protein expression during rat lung carcinogenesis induced by 3-methylcholanthrene and diethylnitrosamine

The epigenetic mechanisms underlying the tumorigenesis caused by polycyclic aromatic hydrocarbons and nitrosamine compounds such as 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are currently unknown. We reported previously that dynamic changes in DNA methylation occurred during MCA/DEN-induced rat lung carcinogenesis. Here, we used the same animal model to further study the evolution of methylation alterations in tumor suppressor genes (TSGs) DAPK1, FHIT, RASSF1A, and SOCS-3. We found that none of these genes were methylated in either normal or hyperplasia tissue. However, as the severity of the cancer progressed through squamous metaplasia and dysplasia to carcinoma in situ (CIS) and infiltrating carcinoma, so methylation became more prevalent. Particularly dramatic increases in the level of methylation, the average number of methylated genes, and the incidence of concurrent methylation in three genes were observed in CIS and infiltrating carcinoma. Similar but less profound changes were seen in squamous metaplasia and dysplasia. Furthermore, methylation status was closely correlated to loss of protein expression for these genes, with protein levels markedly declining along the continuum of carcinogenesis. These results suggest that progressive CpG island hypermethylation leading to inactivation of TSGs might be a vital molecular mechanism in the pathogenesis of MCA/DEN-induced multistep rat lung carcinogenesis.

Distinct Epigenetic Domains Separated by a CTCF Bound Insulator between the Tandem Genes, BLU and RASSF1A

BackgroundTumor suppressor gene (TSG) RASSF1A and candidate TSG BLU are two tandem head-to-tail genes located at 3p21.3. We hypothesized that there may be a concordance on their gene expression and promoter methylation status. If not, then there may be an insulator located between RASSF1A and BLU genes that provides a barrier activity.Methodology/Principal FindingsWe first identified potential transcriptionally important CpG sites using the methylation-specific oligonucleotide array in relation to mRNA expression of RASSF1A and BLU genes in primary lung tumors. We demonstrated that E2F1 bound to the potential transcriptionally important CpG sites in RASSF1A gene of a normal lung cell line expressing RASSF1A transcripts, whereas loss of E2F1 binding to RASSF1A in A549 cancer cell line was the result of DNA methylation. Both RASSF1A and BLU genes had their own potential transcriptionally important CpG regions. However, there was no correlation of methylation status between RASSF1A and BLU. Using gel shift assay and chromatin immunoprecipitation-PCR (ChIP-PCR), we found that CCCTC-binding factor (CTCF) bound to insulator sequences located between these two genes. Bisulfite sequencing and ChIP-PCR revealed distinct methylation and chromatin boundaries separated by the CTCF binding domains in normal cells, whereas such distinct epigenetic domains were not observed in cancer cells. Note that demethylation reagent and histone deacetylase inhibitor treatments led to CTCF binding and recovery of barrier effect for RASSF1A and BLU genes in cancer cells.Conclusions/SignificanceOur study dissects the potential transcriptionally important CpG sites for RASSF1A and BLU genes at the sequence level and demonstrates that CTCF binding to the insulator of BLU gene provides a barrier activity within separate epigenetic domains of the juxtaposed BLU and RASSF1A loci in the 3p21.3 gene cluster region.