Impact of Natural Compounds on DNA Methylation Levels of the Tumor Suppressor Gene RASSF1A in Cancer.

Epigenetic inactivation of tumor suppressor genes (TSG) is a fundamental event in the pathogenesis of human cancer. This silencing is accomplished by aberrant chromatin modifications including DNA hypermethylation of the gene promoter. One of the most frequently hypermethylated TSG in human cancer is the Ras Association Domain Family 1A (RASSF1A) gene. Aberrant methylation of RASSF1A has been reported in melanoma, sarcoma and carcinoma of different tissues. RASSF1A hypermethylation has been correlated with tumor progression and poor prognosis. Reactivation of epigenetically silenced TSG has been suggested as a therapy in cancer treatment. In particular, natural compounds isolated from herbal extracts have been tested for their capacity to induce RASSF1A in cancer cells, through demethylation. Here, we review the treatment of cancer cells with natural supplements (e.g., methyl donors, vitamins and polyphenols) that have been utilized to revert or prevent the epigenetic silencing of RASSF1A. Moreover, we specify pathways that were involved in RASSF1A reactivation. Several of these compounds (e.g., reseveratol and curcumin) act by inhibiting the activity or expression of DNA methyltransferases and reactive RASSF1A in cancer. Thus natural compounds could serve as important agents in tumor prevention or cancer therapy. However, the exact epigenetic reactivation mechanism is still under investigation.

The dual specificity phosphatase 2 gene is hypermethylated in human cancer and regulated by epigenetic mechanisms.

BACKGROUND: Dual specificity phosphatases are a class of tumor-associated proteins involved in the negative regulation of the MAP kinase pathway. Downregulation of the dual specificity phosphatase 2 (DUSP2) has been reported in cancer. Epigenetic silencing of tumor suppressor genes by abnormal promoter methylation is a frequent mechanism in oncogenesis. It has been shown that the epigenetic factor CTCF is involved in the regulation of tumor suppressor genes. METHODS: We analyzed the promoter hypermethylation of DUSP2 in human cancer, including primary Merkel cell carcinoma by bisulfite restriction analysis and pyrosequencing. Moreover we analyzed the impact of a DNA methyltransferase inhibitor (5-Aza-dC) and CTCF on the epigenetic regulation of DUSP2 by qRT-PCR, promoter assay, chromatin immuno-precipitation and methylation analysis. RESULTS: Here we report a significant tumor-specific hypermethylation of DUSP2 in primary Merkel cell carcinoma (p = 0.05). An increase in methylation of DUSP2 was also found in 17 out of 24 (71%) cancer cell lines, including skin and lung cancer. Treatment of cancer cells with 5-Aza-dC induced DUSP2 expression by its promoter demethylation, Additionally we observed that CTCF induces DUSP2 expression in cell lines that exhibit silencing of DUSP2. This reactivation was accompanied by increased CTCF binding and demethylation of the DUSP2 promoter. CONCLUSIONS: Our data show that aberrant epigenetic inactivation of DUSP2 occurs in carcinogenesis and that CTCF is involved in the epigenetic regulation of DUSP2 expression.

Aberrant DNA Methylation of rDNA and PRIMA1 in Borderline Personality Disorder.

Borderline personality disorder (BPD) is a serious psychic disease with a high risk for suicide. DNA methylation is a hallmark for aberrant epigenetic regulation and could be involved in the etiology of BPD. Previously, it has been reported that increased DNA methylation of neuropsychiatric genes is found in the blood of patients with BPD compared to healthy controls. Here, we analyzed DNA methylation patterns of the ribosomal RNA gene (rDNA promoter region and 5'-external transcribed spacer/5'ETS) and the promoter of the proline rich membrane anchor 1 gene (PRIMA1) in peripheral blood samples of 24 female patients (mean age (33 ± 11) years) diagnosed with DSM-IV BPD and in 11 female controls (mean age (32 ± 7) years). A significant aberrant methylation of rDNA and PRIMA1 was revealed for BPD patients using pyrosequencing. For the promoter of PRIMA1, the average methylation of six CpG sites was 1.6-fold higher in BPD patients compared to controls. In contrast, the methylation levels of the rDNA promoter region and the 5'ETS were significantly lower (0.9-fold) in patients with BPD compared to controls. Thus, for nine CpGs located in the rDNA promoter region and for four CpGs at the 5'ETS decreased methylation was found in peripheral blood of patients compared to controls. Our results suggest that aberrant methylation of rDNA and PRIMA1 is associated with the pathogenesis of BPD.

Validation of Tuba1a as appropriate internal control for normalization of gene expression analysis during mouse lung development.

The expression ratio between the analysed gene and an internal control gene is the most widely used normalization method for quantitative RT-PCR (qRT-PCR) expression analysis. The ideal reference gene for a specific experiment is the one whose expression is not affected by the different experimental conditions tested. In this study, we validate the applicability of five commonly used reference genes during different stages of mouse lung development. The stability of expression of five different reference genes (Tuba1a, Actb Gapdh, Rn18S and Hist4h4) was calculated within five experimental groups using the statistical algorithm of geNorm software. Overall, Tuba1a showed the least variability in expression among the different stages of lung development, while Hist4h4 and Rn18S showed the maximum variability in their expression. Expression analysis of two lung specific markers, surfactant protein C (SftpC) and Clara cell-specific 10 kDA protein (Scgb1a1), normalized to each of the five reference genes tested here, confirmed our results and showed that incorrect reference gene choice can lead to artefacts. Moreover, a combination of two internal controls for normalization of expression analysis during lung development will increase the accuracy and reliability of results.

Epigenetically silenced apoptosis-associated tyrosine kinase (AATK) facilitates a decreased expression of Cyclin D1 and WEE1, phosphorylates TP53 and reduces cell proliferation in a kinase-dependent manner.

Silencing of the Apoptosis associated Tyrosine Kinase gene (AATK) has been described in cancer. In our study, we specifically investigated the epigenetic inactivation of AATK in pancreatic adenocarcinoma, lower grade glioma, lung, breast, head, and neck cancer. The resulting loss of AATK correlates with impaired patient survival. Inhibition of DNA methyltransferases (DNMTs) reactivated AATK in glioblastoma and pancreatic cancer. In contrast, epigenetic targeting via the CRISPR/dCas9 system with either EZH2 or DNMT3A inhibited the expression of AATK. Via large-scale kinomic profiling and kinase assays, we demonstrate that AATK acts a Ser/Thr kinase that phosphorylates TP53 at Ser366. Furthermore, whole transcriptome analyses and mass spectrometry associate AATK expression with the GO term 'regulation of cell proliferation'. The kinase activity of AATK in comparison to the kinase-dead mutant mediates a decreased expression of the key cell cycle regulators Cyclin D1 and WEE1. Moreover, growth suppression through AATK relies on its kinase activity. In conclusion, the Ser/Thr kinase AATK represses growth and phosphorylates TP53. Furthermore, expression of AATK was correlated with a better patient survival for different cancer entities. This data suggests that AATK acts as an epigenetically inactivated tumor suppressor gene.

Interferon Regulatory Factor 9 Promotes Lung Cancer Progression via Regulation of Versican.

Transcription factors can serve as links between tumor microenvironment signaling and oncogenesis. Interferon regulatory factor 9 (IRF9) is recruited and expressed upon interferon stimulation and is dependent on cofactors that exert in tumor-suppressing or oncogenic functions via the JAK-STAT pathway. IRF9 is frequently overexpressed in human lung cancer and is associated with decreased patient survival; however, the underlying mechanisms remain to be elucidated. Here, we used stably transduced lung adenocarcinoma cell lines (A549 and A427) to overexpress or knockdown IRF9. Overexpression led to increased oncogenic behavior in vitro, including enhanced proliferation and migration, whereas knockdown reduced these effects. These findings were confirmed in vivo using lung tumor xenografts in nude mice, and effects on both tumor growth and tumor mass were observed. Using RNA sequencing, we identified versican (VCAN) as a novel downstream target of IRF9. Indeed, IRF9 and VCAN expression levels were found to be correlated. We showed for the first time that IRF9 binds at a newly identified response element in the promoter region of VCAN to regulate its transcription. Using an siRNA approach, VCAN was found to enable the oncogenic properties (proliferation and migration) of IRF9 transduced cells, perhaps with CDKN1A involvement. The targeted inhibition of IRF9 in lung cancer could therefore be used as a new treatment option without multimodal interference in microenvironment JAK-STAT signaling.

The RASSF proteins in cancer; from epigenetic silencing to functional characterization.

The Ras-Association Domain Family (RASSF) comprises ten members, termed RASSF1 to RASSF10. RASSF1 to RASSF6 harbor a C-terminal Ras-association (RA) domain and RASSF7 to RASSF10 contain an N-terminal RA domain. Interestingly, it was observed that in various tumor types distinct RASSFs transcripts (e.g. RASSF1A and RASSF2A) are missing due to hypermethylation of their CpG island promoter. Since methylation of the RASSF1A promoter is described as an early and frequent event in tumorigenesis, RASSF1A could serve as a useful diagnostic marker in cancer screens. RASSFs are implicated in various cellular mechanisms including apoptosis, cell cycle control and microtubule stabilization, though little is known about the underlying mechanisms. Tumor suppressing functions were reported for several members. Here we review the current literature on RASSF members focusing on structural, functional and epigenetic aspects. Characterizing the cellular mechanisms that regulate the signaling pathways RASSFs are involved in, could lead to a deeper understanding of tumor development and, furthermore, to new strategies in cancer treatment.

Frequent epigenetic inactivation of RASSF2 in thyroid cancer and functional consequences.

BACKGROUND: The Ras association domain family (RASSF) encodes for distinct tumor suppressors and several members are frequently silenced in human cancer. In our study, we analyzed the role of RASSF2, RASSF3, RASSF4, RASSF5A, RASSF5C and RASSF6 and the effectors MST1, MST2 and WW45 in thyroid carcinogenesis. RESULTS: Frequent methylation of the RASSF2 and RASSF5A CpG island promoters in thyroid tumors was observed. RASSF2 was methylated in 88% of thyroid cancer cell lines and in 63% of primary thyroid carcinomas. RASSF2 methylation was significantly increased in primary thyroid carcinoma compared to normal thyroid, goiter and follicular adenoma (0%, 17% and 0%, respectively; p < 0.05). Patients which were older than 60 years were significantly hypermethylated for RASSF2 in their primary thyroid tumors compared to those younger than 40 years (90% vs. 38%; p < 0.05). RASSF2 promoter hypermethylation correlated with its reduced expression and treatment with a DNA methylation inhibitor reactivated RASSF2 transcription. Over-expression of RASSF2 reduced colony formation of thyroid cancer cells. Functionally our data show that RASSF2 interacts with the proapoptotic kinases MST1 and MST2 and induces apoptosis in thyroid cancer cell lines. Deletion of the MST interaction domain of RASSF2 reduced apoptosis significantly (p < 0.05). CONCLUSION: These results suggest that RASSF2 encodes a novel epigenetically inactivated candidate tumor suppressor gene in thyroid carcinogenesis.

RASSF10 is frequently epigenetically inactivated in kidney cancer and its knockout promotes neoplasia in cancer prone mice.

Kidney cancer incidences are rising globally, thereby fueling the demand for targeted therapies and precision medicine. In our previous work, we have identified and characterized the Ras-Association Domain Family encoding ten members that are often aberrantly expressed in human cancers. In this study, we created and analyzed the Rassf10 knockout mice. Here we show that Rassf10 haploinsufficiency promotes neoplasia formation in two established mouse cancer models (Rassf1A-/- and p53-/-). Haploinsufficient Rassf10 knockout mice were significantly prone to various diseases including lymphoma (Rassf1A-/- background) and thymoma (p53-/- background). Especially Rassf10-/- and p53-deficient mice exhibited threefold increased rates of kidney cysts compared with p53-/- controls. Moreover, we observed that in human kidney cancer, RASSF10 is frequently epigenetically inactivated by its CpG island promoter hypermethylation. Primary tumors of renal clear cell and papillary cell carcinoma confirmed that RASSF10 methylation is associated with decreased expression in comparison to normal kidney tissue. In independent data sets, we could validate that RASSF10 inactivation clinically correlated with decreased survival and with progressed disease state of kidney cancer patients and polycystic kidney size. Functionally, we revealed that the loss of Rassf10 was significantly associated with upregulation of KRAS signaling and MYC expression. In summary, we could show that Rassf10 functions as a haploinsufficient tumor suppressor. In combination with other markers, RASSF10 silencing can serve as diagnostic and prognostic cancer biomarker in kidney diseases.

Epigenetic Inactivation of the Tumor Suppressor IRX1 Occurs Frequently in Lung Adenocarcinoma and Its Silencing Is Associated with Impaired Prognosis.

Iroquois homeobox (IRX) encodes members of homeodomain containing genes which are involved in development and differentiation. Since it has been reported that the IRX1 gene is localized in a lung cancer susceptibility locus, the epigenetic regulation and function of IRX1 was investigated in lung carcinogenesis. We observed frequent hypermethylation of the IRX1 promoter in non-small cell lung cancer (NSCLC) compared to small cell lung cancer (SCLC). Aberrant IRX1 methylation was significantly correlated with reduced IRX1 expression. In normal lung samples, the IRX1 promoter showed lower median DNA methylation levels (<10%) compared to primary adenocarcinoma (ADC, 22%) and squamous cell carcinoma (SQCC, 14%). A significant hypermethylation and downregulation of IRX1 was detected in ADC and SQCC compared to matching normal lung samples (p < 0.0001). Low IRX1 expression was significantly correlated with impaired prognosis of ADC patients (p = 0.001). Reduced survival probability was also associated with higher IRX1 promoter methylation (p = 0.02). Inhibition of DNA methyltransferase (DNMT) activity reactivated IRX1 expression in human lung cancer cell lines. Induced DNMT3A and EZH2 expression was correlated with downregulation of IRX1. On the cellular level, IRX1 exhibits nuclear localization and expression of IRX1 induced fragmented nuclei in cancer cells. Localization of IRX1 and induction of aberrant nuclei were dependent on the presence of the homeobox of IRX1. By data mining, we showed that IRX1 is negatively correlated with oncogenic pathways and IRX1 expression induces the proapoptotic regulator BAX. In conclusion, we report that IRX1 expression is significantly associated with improved survival probability of ADC patients. IRX1 hypermethylation may serve as molecular biomarker for ADC diagnosis and prognosis. Our data suggest that IRX1 acts as an epigenetically regulated tumor suppressor in the pathogenesis of lung cancer.

Frequent occurrence of RASSF1A promoter hypermethylation and Merkel cell polyomavirus in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is one of the most aggressive cancers of the skin. It has recently been reported that integration of a Merkel cell polyomavirus (MCPyV) in receptor tyrosine phosphates type G (PTPRG) gene occurs in MCC, and that viral infections are associated with epigenetic silencing of tumor suppressor genes (TSG) in cancer. To examine whether a correlation between TSG inactivation and viral infection can be found in MCC, we investigated the promoter hypermethylation of RASSF1A, TP73, PTPRG, FHIT, and CDKN2A and the presence of MCPyV and SV40 in 98 MCC by PCR. Hypermethylation of RASSF1A was frequently found in 42 of 83 (51%) of MCC. Methylation of CDKN2A was present in 9 of 41 (22%) of MCC. Hypermethylation of TP73 (0%), PTPRG (4%), and FHIT (0%) was infrequent in MCC. Interestingly, MCPyV was found in 90 of 98 (92%) MCC, however, no SV40 signal was detected. No correlation between TSG hypermethylation and viral infection was found. Our results show frequent hypermethylation of RASSF1A and the presence of MCPyV in primary MCC, and that these events may contribute to the pathogenesis of MCC.

RASSF10 Is a TGFß-Target That Regulates ASPP2 and E-Cadherin Expression and Acts as Tumor Suppressor That Is Epigenetically Downregulated in Advanced Cancer.

The Ras Association Domain Family (RASSF) encodes members of tumor suppressor genes which are frequently inactivated in human cancers. Here, the function and the regulation of RASSF10, that contains a RA (Ras-association) and two coiled domains, was investigated. We utilized mass spectrometry and immuno-precipitation to identify interaction partners of RASSF10. Additionally, we analyzed the up- and downstream pathways of RASSF10 that are involved in its tumor suppressive function. We report that RASSF10 binds ASPP1 (Apoptosis-stimulating protein of p53) and ASPP2 through its coiled-coils. Induction of RASSF10 leads to increased protein levels of ASPP2 and acts negatively on cell cycle progression. Interestingly, we found that RASSF10 is a target of the EMT (epithelial mesenchymal transition) driver TGFß (Transforming growth factor beta) and that negatively associated genes of RASSF10 are significantly over-represented in an EMT gene set collection. We observed a positive correlation of RASSF10 expression and E-cadherin that prevents EMT. Depletion of RASSF10 by CRISPR/Cas9 technology induces the ability of lung cancer cells to proliferate and to invade an extracellular matrix after TGFß treatment. Additionally, knockdown of RASSF10 or ASPP2 induced constitutive phosphorylation of SMAD2 (Smad family member 2). Moreover, we found that epigenetic reduction of RASSF10 levels correlates with tumor progression and poor survival in human cancers. Our study indicates that RASSF10 acts a TGFß target gene and negatively regulates cell growth and invasion through ASPP2. This data suggests that epigenetic loss of RASSF10 contributes to tumorigenesis by promoting EMT induced by TGFß.

The tumor suppressor RASSF1A induces the YAP1 target gene ANKRD1 that is epigenetically inactivated in human cancers and inhibits tumor growth.

The Hippo pathway regulates organ size, growth and comprises several tumor related factors, including the oncoprotein YAP1 and the tumor suppressor RASSF1A. RASSF1A is frequently epigenetically inactivated in cancer. In our study, we analyzed the effect of RASSF1A on the function of YAP1. Expression of YAP1 resulted in the downregulation of several tumor suppressor genes and induction of S-phase. Co-expression with RASSF1A normalized the expression levels of these tumor suppressors and induced a G0-G1 arrest and apoptosis. This effect was associated with the reduction of MDM2 and the increase of p53. These data suggest that the tumor suppressor RASSF1A inhibits the oncogenic potential of YAP1. Additionally, we could show that ANKRD1 is a YAP1 target gene that is induced by RASSF1A. Further analysis revealed that ANKRD1 is epigenetically inactivated in human cancer. ANKRD1 expression induced the expression of TP53 as well as BAX and CDKN1A and reduced colony formation of cancer cells. We found that ANKRD1 interacts with p53 and is involved in the destabilization of MDM2. Additionally, our data indicate that the tumor-suppressive effect of ANKRD1 depends on the presence of p53. These results suggest that ANKRD1 is a tumor-suppressive downstream target of the Hippo pathway that is epigenetically silenced in human cancer.

ZAR1 is a novel epigenetically inactivated tumour suppressor in lung cancer.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths with 1.8 million new cases each year and poor 5-year prognosis. Promoter hypermethylation of tumour suppressors leads to their inactivation and thereby can promote cancer development and progression. RESULTS: In this study, we analysed ZAR1 (zygote arrest 1), which has been said to be a maternal-effect gene and its expression mostly limited to certain reproductive tissues. Our study shows that ZAR1 is expressed in normal lung but inactivated by promoter methylation in lung cancer. ZAR1 is hypermethylated in primary lung cancer samples (22% small cell lung carcinoma (SCLC) and 76% non-small cell lung carcinoma (NSCLC), p < 0.001) vs. normal control lung tissue (11%). In lung cancer cell lines, ZAR1 was significantly methylated in 75% of SCLC and 83% of NSCLC vs. normal tissue (p < 0.005/0.05). In matching tumours and control tissues, we observed that NSCLC primary tumour samples exhibited a tumour-specific promoter methylation of ZAR1 in comparison to the normal control lung tissue. Demethylation treatment of various lung cancer cell lines reversed ZAR1 promoter hypermethylation and subsequently re-established ZAR1 expression. In addition, we could show the growth inhibitory potential of ZAR1 in lung cancer cell lines and cancer cell lines. Exogenous expression of ZAR1 not only inhibited colony formation but also blocked cell cycle progression of cancer cell lines. CONCLUSIONS: Our study shows for the first time the lung tumour-specific epigenetic inactivation of ZAR1 due to DNA methylation of its CpG island promoter. Furthermore, ZAR1 was characterised by the ability to block tumour growth through the inhibition of cell cycle progression in cancer cell lines. We propose that ZAR1 could serve as an epigenetically inactivated biomarker in lung cancer.

Epigenetic silencing of downstream genes mediated by tandem orientation in lung cancer.

Epigenetic deregulation is of importance in tumorigenesis. In particular CpG islands (CGI), are frequently hypermethylated. Here, genome-wide DNA-methylation profiles of 480,000 CpGs in lung cancer cells were generated. It was observed that intra- and intergenic CGI exhibited higher methylation compared to normal cells. The functional annotation of hypermethylated CGI revealed that the hypermethylation was associated with homeobox domain genes and targets marked by repressive histone modifications. The strongest methylation variation was observed in transitional areas of CGI, termed shores. 5'-shores of promoter-associated CGI in lung cancer cell lines were higher methylated than 3'-shores. Within two tandem-oriented genes, a significant hypermethylation of the downstream-located CGI promoters was revealed. Hypermethylation correlates with the length of the intergenic region between such tandem genes. As the RASSF1A tumor suppressor gene represents such a downstream tandem gene, its silencing was analyzed using an inducible system. It was determined that the induction of an upstream gene led to a repression of RASSF1A through a process involving histone deacetylases and CPSF1. A tumor-specific increase in expression of histone deacetylases and CPSF1 was detected in lung cancer. Our results suggest that the downstream gene could be susceptible to epigenetic silencing when organized in a tandem orientation.

Aberrant Promoter Methylation of the Tumour Suppressor RASSF10 and Its Growth Inhibitory Function in Breast Cancer.

Breast cancer is the most common cancer in women, with 1.7 million new cases each year. As early diagnosis and prognosis are crucial factors in cancer treatment, we investigated potential DNA methylation biomarkers of the tumour suppressor family Ras-association domain family (RASSF). Promoter hypermethylation of tumour suppressors leads to their inactivation and thereby promotes cancer development and progression. In this study we analysed the tumour suppressors RASSF1A and RASSF10. Our study shows that RASSF10 is expressed in normal breast but inactivated by methylation in breast cancer. We observed a significant inactivating promoter methylation of RASSF10 in primary breast tumours. RASSF10 is inactivated in 63% of primary breast cancer samples but only 4% of normal control breast tissue is methylated (p < 0.005). RASSF1A also shows high promoter methylation levels in breast cancer of 56% vs. 8% of normal tissue (p < 0.005). Interestingly more than 80% of breast cancer samples harboured a hypermethylation of RASSF10 and/or RASSF1A promoter. Matching samples exhibited a strong tumour specific promoter methylation of RASSF10 in comparison to the normal control breast tissue. Demethylation treatment of breast cancer cell lines MCF7 and T47D reversed RASSF10 promoter hypermethylation and re-established RASSF10 expression. In addition, we could show the growth inhibitory potential of RASSF10 in breast cancer cell lines MCF7 and T47D upon exogenous expression of RASSF10 by colony formation. We could further show, that RASSF10 induced apoptotic changes in MCF7 and T47D cells, which was verified by a significant increase in the apoptotic sub G1 fraction by 50% using flow cytometry for MCF7 cells. In summary, our study shows the breast tumour specific inactivation of RASSF10 and RASSF1A due to DNA methylation of their CpG island promoters. Furthermore RASSF10 was characterised by the ability to block growth of breast cancer cell lines by apoptosis induction.

Non-invasive lung cancer diagnosis by detection of GATA6 and NKX2-1 isoforms in exhaled breath condensate.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Early LC diagnosis is crucial to reduce the high case fatality rate of this disease. In this case-control study, we developed an accurate LC diagnosis test using retrospectively collected formalin-fixed paraffin-embedded (FFPE) human lung tissues and prospectively collected exhaled breath condensates (EBCs). Following international guidelines for diagnostic methods with clinical application, reproducible standard operating procedures (SOP) were established for every step comprising our LC diagnosis method. We analyzed the expression of distinct mRNAs expressed from GATA6 and NKX2-1, key regulators of lung development. The Em/Ad expression ratios of GATA6 and NKX2-1 detected in EBCs were combined using linear kernel support vector machines (SVM) into the LC score, which can be used for LC detection. LC score-based diagnosis achieved a high performance in an independent validation cohort. We propose our method as a non-invasive, accurate, and low-price option to complement the success of computed tomography imaging (CT) and chest X-ray (CXR) for LC diagnosis.

Claudin11 Promoter Hypermethylation Is Frequent in Malignant Melanoma of the Skin, but Uncommon in Nevus Cell Nevi.

Epigenetic inactivation of tumor-related genes is an important characteristic in the pathology of human cancers, including melanomagenesis. We analyzed the epigenetic inactivation of Claudin 11 (CLDN11) in malignant melanoma (MM) of the skin, including six melanoma cell lines, 39 primary melanoma, 41 metastases of MM and 52 nevus cell nevi (NCN). CLDN11 promoter hypermethylation was found in 19 out of 39 (49%) of the primary MM and in 21 out of 41 (51%) of the MM metastases, but only in eight out of 52 (15%) of NCN (p = 0.001 and p = 0.0003, respectively). Moreover, a significant increase in the methylation level of CLDN11 from primary melanomas to MM metastases was revealed (p = 0.003). Methylation of CLDN11 was significantly more frequent in skin metastases (79%) compared to brain metastases (31%; p = 0.007). CLDN11 methylation was also found in five out of six MM cell lines (83%) and its promoter hypermethylation correlated with a reduced expression. Treatment of MM cell lines with a DNA methylation inhibitor reactivated CLDN11 transcription by its promoter demethylation. In summary, CLDN11 proved to be an epigenetically inactivated tumor related gene in melanomagenesis, and analysis of CLDN11 methylation level represents a potential tool for assisting in the discrimination between malignant melanoma and nevus cell nevi.

Promoter methylation status of Ras-association domain family members in pheochromocytoma.

Pheochromocytomas (PCCs) are rare neuroendocrine tumors that arise from the medulla of the adrenal gland or the sympathetic ganglia and are characterized by the secretion of catecholamines. In 30-40% of patients, PCCs are genetically determined by susceptibility genes as various as RET, VHL, and NF1. We have analyzed the Ras-association domain family members (RASSFs) in PCCs regarding their inactivating promoter hypermethylation status. Previously, we reported a promoter methylation in PCC for the first family member RASSF1A. Promoter hypermethylation of CpG islands leads to the silencing of the according transcript and is a common mechanism for inactivation of tumor suppressors. In this study, we observed inactivating DNA modifications for the RASSF members RASSF2, RASSF5A, RASSF9, and RASSF10, but not for the members RASSF3, RASSF4, RASSF5C, RASSF6, RASSF7, and RASSF8. The degree of promoter methylation was 19% for RASSF2, 67% for RASSF5A, 18% for RASSF9, and 74% for RASSF10. Interestingly, the degree of hypermethylation for RASSF10 in hereditary PCCs was 89 vs. 60% in sporadic PCCs. A similar but less dramatic effect was observed in RASSF5A and RASSF9. Including all RASSF members, we found that of 25 PCCs, 92% show promoter methylation in at least in one RASSF member. In 75% of the hereditary PCC samples, we found two or more methylated RASSF promoters, whereas in sporadic PCCs only 46% were observed. In summary, we could show that in PCC several RASSF members are strongly hypermethylated in their promoter regions and methylation of more than one RASSF member occurs in the majority of PCCs. This adds the inactivation of genes of the RASSF tumor suppressor family to the already known deregulated genes of PCC.

The apoptosis associated tyrosine kinase gene is frequently hypermethylated in human cancer and is regulated by epigenetic mechanisms.

Epigenetic gene inactivation through promoter hypermethylation is an important aberration involved in the silencing of tumor-associated genes in cancer. Here we identified the apoptosis associated tyrosine kinase (AATK) as an epigenetically downregulated tumor related gene. We analyzed the epigenetic regulation of AATK in several human cancer cell lines and normal tissues by methylation and expression analysis. Hypermethylation of AATK was also analyzed in 25 primary lung tumors, 30 breast cancers and 24 matching breast tissues. In normal tissues the AATK CpG island promoter was unmethylated and AATK was expressed. Hypermethylation of AATK occurred frequently in 13 out of 14 (93%) human cancer cell lines. Methylation was reversed by 5-aza-2'-deoxycytidine treatment leading to re-expression of AATK in cancer cell lines. Aberrant methylation of AATK was also revealed in primary lung (40%) and breast (53%) cancers, but was found to be significantly less methylated in matching normal breast tissues (17%; p<0.01). In addition, we observed that AATK is epigenetically reactivated through the chromatin regulator CTCF. We further show that overexpression of Aatk significantly suppresses colony formation in cancer cell lines. Our findings suggest that the apoptosis associated tyrosine kinase is frequently inactivated in human cancers and acts as a tumor suppressive gene.