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1.  Reassembly of Nucleosomes at the MLH1 Promoter Initiates Resilencing Following Decitabine Exposure 
PLoS Genetics  2013;9(7):e1003636.
Hypomethylating agents reactivate tumor suppressor genes that are epigenetically silenced in cancer. Inevitably these genes are resilenced, leading to drug resistance. Using the MLH1 tumor suppressor gene as a model, we showed that decitabine-induced re-expression was dependent upon demethylation and eviction of promoter nucleosomes. Following decitabine withdrawal, MLH1 was rapidly resilenced despite persistent promoter demethylation. Single molecule analysis at multiple time points showed that gene resilencing was initiated by nucleosome reassembly on demethylated DNA and only then was followed by remethylation and stable silencing. Taken together, these data establish the importance of nucleosome positioning in mediating resilencing of drug-induced gene reactivation and suggest a role for therapeutic targeting of nucleosome assembly as a mechanism to overcome drug resistance.
Author Summary
Hypomethylating agents are emerging as effective cancer therapies. However, their therapeutic effects are transient and drug resistance inevitably develops. While resistance is associated with resilencing of genes initially demethylated by the drug, the mechanism underlying this resilencing is unknown. We provide evidence that the rapid reassembly of nucleosomes at transcription start sites initiates resilencing and is a prerequisite for promoter remethylation. This finding shows reassembly of nucleosomes at the promoter of critical genes is a potential early marker of resistance to hypomethylating agents. Our findings have implications for the treatment of cancer using epigenetic therapies that target DNA methylation alone, and suggest that overcoming drug resistance will require therapeutic strategies which prevent nucleosome deposition.
doi:10.1371/journal.pgen.1003636
PMCID: PMC3723495  PMID: 23935509
2.  A reinvestigation of somatic hypermethylation at the PTEN CpG island in cancer cell lines 
Background
PTEN is an important tumour suppressor gene that is mutated in Cowden syndrome as well as various sporadic cancers. CpG island hypermethylation is another route to tumour suppressor gene inactivation, however, the literature regarding PTEN hypermethylation in cancer is controversial. Furthermore, investigation of the methylation status of the PTEN CpG island is challenging due to sequence homology with the PTEN pseudogene, PTENP1. PTEN shares a CpG island promoter with another gene known as KLLN. Here we present a thorough reinvestigation of the methylation status of the PTEN CpG island in DNA from colorectal, breast, ovarian, glioma, lung and haematological cancer cell lines.
Results
Using a range of bisulphite-based PCR assays we investigated 6 regions across the PTEN CpG island. We found that regions 1-4 were not methylated in cancer cell lines (0/36). By allelic bisulphite sequencing and pyrosequencing methylation was detected in regions 5 and 6 in colorectal, breast and haematological cancer cell lines. However, methylation detected in this region was associated with the PTENP1 promoter and not the PTEN CpG island.
Conclusions
We show that methylation of the PTEN CpG island is a rare event in cancer cell lines and that apparent methylation most likely originates from homologous regions of the PTENP1 pseudogene promoter. Future studies should utilize assays that reliably discriminate between PTEN and PTENP1 to avoid data misinterpretation.
doi:10.1186/1480-9222-14-5
PMCID: PMC3342897  PMID: 22490388
DNA methylation; Epigenetic; PTEN; KILLIN; PTENP1; Pseudogene; Cowden syndrome
3.  Nuclear localisation and epigenetic inactivation of the ras effector/tumour suppressor RASSF2A in multiple human cancers 
Oncogene  2007;27(12):1805-1811.
RASSF2 is a recently identified member of a class of novel tumour suppressor genes, all containing a ras association domain. We previously demonstrated that the A isoform of RASSF2, is frequently inactivated by promoter region hypermethylation in colorectal tumours and adenomas, methylation was tumour specific and that expression in methylated tumour lines could be reactivated by treatment with 5-aza-2dc. RASSF2 resides at 20p13, this region has been demonstrated to be frequently lost in human cancers. In this report we investigated methylation status of the RASSF2A promoter CpG island in a series of breast, ovarian and non-small cell lung cancers (NSCLC). RASSF2A was frequently methylated in breast tumour cell lines 65% (13/20) and in primary breast tumours 38% (15/40). RASSF2A gene expression could be switched back on in methylated breast tumour cell lines after treatment with 5-aza-2dC, whilst unmethylated lines showed no difference in level of expression before and after 5-aza-2dC treatment. RASSF2A was also frequently methylated in NSCLC tumours 44% (22/50). Methylation in breast tumours and NSCLC was tumour specific. We did not detect RASSF2A methylation in ovarian tumours (0/17). Furthermore no mutations were found in the coding region of RASSF2A in these ovarian tumours.
RASSF2A suppressed breast tumour cell growth in vitro (through colony formation and soft agar assays) and in vivo. We identified a highly conserved putative bipartite nuclear localisation signal (NLS) between amino acids 151 and 167 in the RASSF2A sequence and demonstrated that endogenous RASSF2A localised to the nucleus. Mutation of the putative nuclear localisation signal abolished the nuclear localisation so RASSF2A became predominantly cytoplasmic. Our data indicates that RASSF2A is frequently methylated in colorectal, breast and NSCLC tumours, furthermore, the methylation is tumour specific. Hence we have identified RASSF2A as a novel methylation marker for multiple malignancies and it has the potential to be developed into a valuable marker for screening several cancers in parallel using promoter hypermethylation profiles.
We also demonstrate that RASSF2 has a functional NLS signal. Furthermore this is the first report demonstrating that RASSF2 suppresses growth of cancer cells in vivo. Hence providing further evidence for its role as a tumour suppressor gene located at 20p13.
doi:10.1038/sj.onc.1210805
PMCID: PMC2948550  PMID: 17891178
4.  Identification of 5 novel genes methylated in breast and other epithelial cancers 
Molecular Cancer  2010;9:51.
Background
There are several high throughput approaches to identify methylated genes in cancer. We utilized one such recently developed approach, MIRA (methylated-CpG island recovery assay) combined with CpG island arrays to identify novel genes that are epigenetically inactivated in breast cancer.
Results
Using this approach we identified numerous CpG islands that demonstrated aberrant DNA methylation in breast cancer cell lines. Using a combination of COBRA and sequencing of bisulphite modified DNA, we confirmed 5 novel genes frequently methylated in breast tumours; EMILIN2, SALL1, DBC1, FBLN2 and CIDE-A. Methylation frequencies ranged from between 25% and 63% in primary breast tumours, whilst matched normal breast tissue DNA was either unmethylated or demonstrated a much lower frequency of methylation compared to malignant breast tissue DNA. Furthermore expression of the above 5 genes was shown to be restored following treatment with a demethylating agent in methylated breast cancer cell lines. We have expanded this analysis across three other common epithelial cancers (lung, colorectal, prostate). We demonstrate that the above genes show varying levels of methylation in these cancers. Lastly and most importantly methylation of EMILIN2 was associated with poorer clinical outcome in breast cancer and was strongly associated with estrogen receptor as well as progesterone receptor positive breast cancers.
Conclusion
The combination of the MIRA assay with CpG island arrays is a very useful technique for identifying epigenetically inactivated genes in cancer genomes and can provide molecular markers for early cancer diagnosis, prognosis and epigenetic therapy.
doi:10.1186/1476-4598-9-51
PMCID: PMC2841122  PMID: 20205715
5.  RASSF2 associates with and stabilises the proapoptotic kinase MST2 
Oncogene  2009;28(33):2988-2998.
RASSF2 is a tumour suppressor that in common with the rest of the RASSF family contains Ras association and SARAH domains. We identified the proapoptotic kinases MST1 and MST2 as the most significant binding partners of RASSF2, confirmed the interactions at endogenous levels and demonstrated that RASSF2 immunoprecipitates active MST1/2. We then demonstrated that RASSF2 can be phosphorylated by a co-immunoprecipitating kinase which is likely to be MST1/2. Furthermore, we demonstrated that RASSF2 and MST2 do indeed colocalise, but whilst RASSF2 alone is nuclear, the presence of MST1 or MST2 results in colocalisation in the cytoplasm. Expression of RASSF2 (stably in MCF7 or transiently in HEK-293) increases MST2 levels and knockdown of RASSF2 in HEK-293 cells reduces MST2 levels, additionally colorectal tumour cell lines and primary tumours with low RASSF2 levels show decreased MST2 protein levels. This is likely to be mediated by RASSF2-dependent protection of MST2 against proteolytic degradation. Our findings suggest that MST2 and RASSF2 form an active complex in vivo where RASSF2 is maintained in a phosphorylated state and protects MST2 from degradation and turnover. Thus we propose that the frequent loss of RASSF2 in tumours results in destabilisation of MST2 and thus decreased apoptotic potential.
doi:10.1038/onc.2009.152
PMCID: PMC2829092  PMID: 19525978
RASSF2; MST2; MST1; proteomics; epigenetics
6.  The novel RASSF6 and RASSF10 candidate tumour suppressor genes are frequently epigenetically inactivated in childhood leukaemias 
Molecular Cancer  2009;8:42.
Background
The Ras-assocation family (RASSF) of tumour suppressor genes (TSGs) contains 10 members that encode proteins containing Ras-assocation (RA) domains. Several members of the RASSF family are frequently epigenetically inactivated in cancer, however, their role in leukaemia has remained largely uninvestigated. Also, RASSF10 is a predicted gene yet to be experimentally verified. Here we cloned, characterised and demonstrated expression of RASSF10 in normal human bone marrow. We also determined the methylation status of CpG islands associated with RASSF1–10 in a series of childhood acute lymphocytic leukaemias (ALL) and normal blood and bone marrow samples.
Results
COBRA and bisulphite sequencing revealed RASSF6 and RASSF10 were the only RASSF members with a high frequency of leukaemia-specific methylation. RASSF6 was methylated in 94% (48/51) B-ALL and 41% (12/29) T-ALL, whilst RASSF10 was methylated in 16% (8/51) B-ALL and 88% (23/26) T-ALL. RASSF6 and RASSF10 expression inversely correlated with methylation which was restored by treatment with 5-aza-2'deoxycytidine (5azaDC).
Conclusion
This study shows the hypermethylation profile of RASSF genes in leukaemias is distinct from that of solid tumours and represents the first report of inactivation of RASSF6 or RASSF10 in cancer. These data show epigenetic inactivation of the candidate TSGs RASSF6 and RASSF10 is an extremely frequent event in the pathogenesis of childhood leukaemia. This study also warrants further investigation of the newly identified RASSF member RASSF10 and its potential role in leukaemia.
doi:10.1186/1476-4598-8-42
PMCID: PMC2711046  PMID: 19570220
7.  The Role of RASSF1A Methylation in Cancer 
Disease Markers  2007;23(1-2):73-87.
Tumour suppressor gene inactivation is critical to the pathogenesis of cancers; such loss of function may be mediated by irreversible processes such as gene deletion or mutation. Alternatively tumour suppressor genes may be inactivated via epigenetic processes a reversible mechanism that promises to be more amenable to treatment by therapeutic agents. The CpG dinucleotide is under-represented in the genome, but it is found in clusters within the promoters of some genes, and methylation of these CpG islands play a critical role in the control of gene expression. Inhibitors of the DNA methyltransferases DNMT1 and DNMT3b have been used in a clinical setting, these nucleotide analogues lack specificity but the side effects of low dose treatments were minimal and in 2004 Vidaza (5-azacitidine) was licensed for use in myelodysplastic syndrome. Methylation inhibitors are also entering trials in conjunction with another class of epigenetic modifiers, the histone deacetylase inhibitors and this epigenetic double bullet offers hope of improved treatment regimes. Recently there has been a plethora of reports demonstrating epigenetic inactivation of genes that play important roles in development of cancer, including Ras-association domain family of genes. Epigenetic inactivation of RASSF1A (Ras-association domain family 1, isoform A) is one of the most common molecular changes in cancer. Hypermethylation of the RASSF1A promoter CpG island silences expression of the gene in many cancers including lung, breast, prostate, glioma, neuroblastoma and kidney cancer. Several recent studies have illustrated the diagnostic and prognostic potential of RASSF1A methylation. This presents RASSF1A methylation as an attractive biomarker for early cancer detection which, for most cancers, results in improved clinical outcome. DNA methylation analysis is applicable to a range of body fluids including serum, urine, bronchioalveolar lavage and sputum. The ease with which these body fluids can be acquired negates the need for invasive procedures to obtain biopsy material. This review will discuss the feasibility of using RASSF1A methylation as a diagnostic and prognostic marker in cancer management.
doi:10.1155/2007/291538
PMCID: PMC3850810  PMID: 17325427
RASSF1A; tumour suppressor gene; 3p21.3; ras association domain; methylation; cancer

Results 1-7 (7)