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The Genomic Data Commons (GDC) Data Portal is an interactive data system for researchers to search, download, upload, and analyze harmonized cancer genomic data sets, including TCGA. Figure 4 : Is cancer triggered by altered signalling of nicotinic acetylcholine receptors?
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Circulating Methylated DNA as Biomarkers for Cancer DetectionHongchuan Jin1, Yanning Ma1, Qi Shen1 and Xian Wang1[1] Department of Medical Oncology, Laboratory of Cancer Epigenetics, Biomedical Research Center, Sir Runrun Shaw Hospital, Zhejiang University, China1.
Phase I study of epigenetic modulation with 5-azacytidine and valproic acid in patients with advanced cancers. Azacitidine for the treatment of patients with acute myeloid leukemia with 20%-30% blasts and multilineage dysplasia. Phase 1b-2a study to reverse platinum resistance through use of a hypomethylating agent, azacitidine, in patients with platinum-resistant or platinum-refractory epithelial ovarian cancer. Continued azacitidine therapy beyond time of first response improves quality of response in patients with higher-risk myelodysplastic syndromes.
Azacitidine favorably modulates PSA kinetics correlating with plasma DNA LINE-1 hypomethylation in men with chemonaive castration-resistant prostate cancer. Molecular and immunological detection of circulating tumor cells and micrometastases from solid tumors.
Detection of circulating tumor cells and of tumor DNA in plasma during tumor progression in rats. Circulating DNA in higher organisms cancer detection brings back to life an ignored phenomenon. The occurrence of single-stranded DNA in the serum of patients with systemic lupus erythematosus and other diseases. Detection of K-ras gene mutations in plasma DNA of patients with pancreatic adenocarcinoma: correlation with clinicopathological features. Kras mutations in DNA extracted from the plasma of patients with pancreatic carcinoma: diagnostic utility and prognostic significance. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.
The nicotinic agonists at the top of the signalling cascades are listed in the order of their affinity to the nAChRs. IntroductionIn addition to genetic alterations including deletion or point mutations, epigenetic changes such as DNA methylation play an important role in silencing tumor suppressor genes during cancer development.
Circulating Tumor cells (CTC) escape from primary sites and spread into the vessel to form metastases in the distal organs with. MSP, BGS and COBRA are based on bisulfite-mediated conversion of unmethylated cytosines into uracils.
By adding a methyl group from S-adenosyl-L-methionine to the cytosine pyrimidine or adenine purine ring, DNA methylation is important to maintain genome structure and regulate gene expression. In vitro, the homomeric and heteromeric nAChRs jointly stimulate the indicated signalling cascades. Cell free DNAs (cf-DNAs) are released into the circulation from dead cancer cells or proliferating tumor cells. CpG methylation could block DNA digestion by some restriction enzymes, making it possible to determine methylation status independent of bisulfite treatment by analyzing digestion products. In mammalian adult tissues, DNA methylation occurs in CpG dinucleotides that often cluster in the genome as CpG islands in the 5’ regulatory regions of the genes. Alternatively, DNA fragments containing methylated CpG sites could be enriched by anti-methylcytosine antibody or methylation binding proteins. Through recruiting transcriptional co-repressors including methyl-CpG-binding domain proteins (MBDs) and chromatin remodeling proteins like histone deacetylases (HDACs) or impeding the binding of transcriptional activators, DNA methylation could suppress the transcription of many tumor suppressor genes critical to cancer initiation and progression [1-3].
Influx of Ca2+ and other cations through the nAChRs and voltage-gated Ca2+ channels triggers the release of epidermal growth factor (EGF), which activates the EGFR signalling cascade.
Advances in next generation genome sequencing technology led to the development of noel techniques such as SMRT which can specially analyze 5-methylcytosines with genome wide coverage. More and more results confirmed that cancer is a multi-stage process fuelled by many epigenetic changes in addition to genetic changes in DNA sequence [4]. The responsiveness of this pathway is enhanced by 7nAChR-mediated activation of Ras through -arrestin-dependent SRC. Chemical molecules like Trichostatin A (TSA) and 5-aza-2'-deoxycytidine (5-Aza-CdR) targeting epigenetic regulators such as histone modifications and DNMTs (DNA methyltransferases) have been found to inhibit tumor growth both in vitro and in vivo. In turn, the EGFR activates the Akt pathway and its downstream effectors, X-linked inhibitor of apoptosis protein (XIAP)–survivin and nuclear factor-B (NF-B). By reversing the epigenetic silencing of important tumor suppressor genes, an increasing number of epigenetic drugs such as 5-Aza-CdR, 5-Aza-CR and Vorinostat (SAHA) are currently investigated in the clinical trials for cancer treatment as a single drug or in combination with other epigenetic drugs or other approaches such as chemotherapy and showed very promising activities by offering significant clinical benefits to cancer patients [5-13].
As one of the major epigenetic changes to inactivate tumor suppressor genes critical to human cancer development, DNA methylation was recognized as the biomarker for cancer detection or outcome prediction in addition to the identification of novel tumor suppressor genes. DNA mutations will occur randomly in any nucleotides of one particular gene and the comprehensive determination of DNA mutations is thus very difficult and time-consuming.

In contrast, aberrant DNA hypermethylation usually takes place in defined CpG Islands within the regulatory region of the genes and it is much more convenient to detect DNA methylation in a quantitatively manner. In addition, DNA methylation can be amplified and is thus easily detectable using PCR-based approaches even when the DNA concentration after sample extraction is relatively low. Due to such advantages over DNA mutation- or protein-based biomarkers, DNA methylation-based biomarkers have been intensively investigated in the recent years.
A large body of research reports has proved the value of DNA methylations in the prognosis prediction and detection of various cancers.
DNAs used for such methylation analyses are usually extracted from tumor tissues harvested after surgical operation or biopsy, thus limiting its wide application as the biomarkers for the early detection or screening of human cancers.
Recently, it has been reported that there are certain amount of circulating DNAs in the peripheral blood of cancer patients, providing an ideal source to identify novel biomarkers for non-invasive detection of cancers. Both genetic and epigenetic changes found in the genomic DNAs extracted from primary tumor cells could be detected in the circulating DNAs, indicating that the detection of methylated DNAs in the circulation represents a new direction to develop novel biomarkers for cancer detection or screening in a non-invasive manner. Cell free DNA in the circulationAccording to the origin of circulating tumor-related DNA, it could be grouped into circulating cell free DNA or DNA from cells in the blood such as circulating tumor cells (CTC) in cancer patients (Figure 1). In 1869, the Australian physician Thomas Ashworth observed CTCs in the blood of a cancer patient. Therefore, it was postulated that CTCs were responsible for the tumor metastases in distal sites and should have important prognostic and therapeutic implications [14-16]. Usually around 1-10 CTCs together with several million blood cells could be found in 1 ml of whole blood, making the specific and sensitive detection of CTCs very difficult [17-18].
Until recently, technologies with the requisite sensitivity and reproducibility for CTC detection have been developed to precisely analyze its biological and clinical relevance. The US Food and Drug Administration (FDA) approved the test for determining CTC levels in patients with metastatic breast cancer in 2004. Currently, it has been expanded to other cancer types such as advanced colorectal cancer and prostate cancer. Although CTCs-counting based test have proven its value in predicting prognosis and monitoring therapeutic effects, the number of CTCs per ml of blood limited its sensitivity greatly [19]. With the development of high-sensitive PCR-based methods, the detection of gene mutations or epigenetic changes such as DNA methylation within small amount of CTCs could be the next generation of CTC-based test for cancer detection. However, the cost of such tests will be greatly exacerbated, thus limiting its wide application in the clinic [20-22]. Early in 1948, two French scientists Mandel and Metais firstly reported the presence of cf-DNAs in human plasma [25]. Such an important discovery has been unnoticed for a long time until cell-free circulating nucleic acid was found to promote the spread and metastasis of crown gall tumor in plants [26].
Subsequently, increased level of cf-DNAs was found in patients with various diseases such as lupus erythematosus and rheumatoid arthritis cancer [27-28]. Moreover, greater amounts of cf-DNA were found in the peripheral blood of cancer patients with tumor metastases and cf-DNA levels decreased dramatically after radiotherapy while persistently high or increasing DNA concentrations were associated with a lack of response to treatment [29], clearly revealing the potential value of cf-DNA as biomarker for cancer detection. Following studies confirmed that cf-DNAs in the plasma contains genetic and epigenetic changes specific to DNAs within the tumor cells from primary tissues, indicating that tumor specific cf-DNAs are originated from tumor cells rather than lymphocytes reacting towards the disease [30-31].
For example, K-Ras mutation was found in cf-DNA from 17 out of 21 patients with pancreatic adenocarcinoma and mutations were similar in corresponding plasma and tissues samples. Importantly, such DNA alterations were found in patients with pancreatitis who were diagnosed as pancreatic cancer 5-14 months later, indicating that release of tumor-specific DNA into the circulation is an early event in cancer development and cf-DNA could be used as the biomarkers for early cancer detection [32]. Treatment resulted in disappearance of K-Ras mutations in plasma DNA in six of nine patients. Three patients with a persistently positive K-Ras gene mutation in plasma samples from patients before and after treatment showed early recurrence or progression and pancreatic carcinoma patients with the mutant-type K-ras gene in plasma DNA exhibited a shorter survival time than patients with the wild-type gene, indicating the cf-DNA could be of value in monitoring disease progression or evaluating treatment response [31, 33]. Through quantitatively analyzing plasma DNAs from patients with organ transplantation, Lo et al found that the majority of plasma DNAs was released from the hematopoietic system. However, donor DNA could be detected in the plasma of recipients suffering from the graft rejection because of the large amount of cell death which promotes the release of donor DNAs into the peripheral blood of the recipients [34]. Therefore, it was postulated that cell-free tumor related DNA could originate from the apoptotic tumor cells since high-rate of apoptosis indeed occurs in primary and metastatic tumor tissues. However, cf-DNA quantities are significantly reduced in cancer patients after radiotherapy when a great number of tumor cells were believed to undergo apoptotic cell death and cf-DNAs in supernatants of cultured cancer cells increases with cell proliferation rather than apoptosis or necrosis, indicating that proliferating tumor cells could actively release cf-DNA into the tumor microenvironment and circulation. In contrast to labile RNAs that were included into the actively secreted exosomes, the nature of cf-DNAs remains to be clarified.
As negatively charged molecules, cf-DNA was bound by plasma proteins to escape from endonuclease-mediated degradation. Meanwhile, secreted exosomes could remodel microenviroments and promote tumor metastasis since RNAs within exosomes especially microRNA with high stability may influence gene expression in neighbor cells. However, it was supposed that cf-DNA could play a role as vaccine in tumor microenvironment. Methods for the detection of methylated DNA It is unclear so far whether serum or plasma is better for cf-DNA extraction. Although the DNA amount is significantly higher in the serum, the majority of the increase was due to the release of nuclear acids from destroyed blood cells during blood clotting [35].

In addition, the time gap between blooding drawing and DNA extraction as well as the methodologies used for DNA isolation contribute greatly to the amount of cf-DNA harvested. Therefore, in order to determine the quantity of potential cf-DNA-based biomarkers precisely and promote its wide application for cancer detection, it is very important to unify the source as well as the methodologies for cf-DNA extraction and use various internal controls to adjust possible inter-laboratory variations. Advances in next generation genome sequencing technology led to the development of noel techniques such as SMRT which can specially analyze 5-methylcytosines with genome wide coverage.In general, the detection of DNA methylation could be bisulfite-dependent or -independent (Figure 2). The following analyses such as methylation-and unmethylation specific polymerase chain reaction (M- and U-SP), bisulfite genome sequencing (BGS) or combined bisulfite restriction analysis (COBRA) could determine the conversion of CpG sites of interest, thus reflecting their methylation status as methylated or unmethylated [37].
Generated from the oxidation of 5-methylcytosine by the Tet family of enzymes, 5-hydoxymethylcytosine was first found in bacteriophages and recently shown to be abundant in human and mouse brains as well as in embryonic stem cells [39-40]. Although the exact relevance of 5-hydoxymethylcytosine in the genome is still not fully clarified, it has been found to regulate gene expression or promote DNA demethylation. The in vitro synthesized artificial oligonucleotides containing 5-hydoxymethylcytosines can be converted into unmodified cytosines when introduced into mammalian cells, indicating that 5-hydoxymethylcytosine might be one of intermediate products during active DNA demethylation [41].
Therefore, the increase of 5-hydoxymethylcytosine might reflect the demethylation of CpG dinucleotides. Unfortunately, 5-hydoxymethylcytosines, similar to 5-methylcytosines, appear to be resistant to bisulfite-mediated conversion and PCR could amplify DNA fragments containing 5-hydoxymethylcytosines or 5-methylcytosines with similar efficiency [42-43].
Therefore, bisulfite-dependent methylation analyses could produce false positive results by counting 5-hydoxymethylcytosines into 5-methylcytosines. In addition to 5-hydroxymethylcytosines, some forms of DNA modifications such as the seventh base, 5-formylcytosine and the eighth base, 5-carboxylcytosine, have been found in mammalian cells recently [44-47].
As the products of 5-hydoxymethylcytosine oxidation through TET hydroxylases, both 5-formylcytosine and 5-carboxylcytosine will be read as the uracil after bisulfite conversion, thus making it impossible for bisulfite-dependent analyses to distinguish unmodified cytosines from 5-formylcytosines and 5-carboxylcytosines.
Bisulfite independent analyses such as MedIP (methylated DNA immunoprecipitation) could more or less detect DNA methylation specifically. In bisulfite independent analyses, 5-methylcytosines are differentiated from unmethylated cytosine by either enzyme digestion or affinity enrichment.
DNA methylation analysis using restriction enzyme digestion is based on the property of some methylation-sensitive and -resistant restriction enzymes such as HpaII and MspI that target CCGG for digestion.
As a primary method to analyze DNA methylation, it can only determine the methylation of CpG in the context of CCGG tetranucleotides and will overlook the majority of CpG dinucleotides in the genome. The development of monoclonal antibody specific to 5-methylcytosines revolutionized the analyses of DNA methylation [48-49]. Immunoprecipitated DNA by this antibody could be subject to DNA microarray or even deep sequencing to reveal novel sequences or sites containing 5-methylcytosines [50].
However, 5-methylcytosines could present not only in CpG dinucleotides but also in CHH or CHG trinucleotides, especially in plants, human embryonic stem cells and probably cancer cells as well. CHH methylation indicates a 5-methylcytosine followed by two nucleotides that may not be guanine and CHG methylation refers to a 5-methylcytosine preceding an adenine, thymine or cytosine base followed by guanine.
Such non-CpG DNA methylations were enriched at transposons and repetitive regions, although the exact biological relevance remains unknown. However, antibody against 5-methylcytosine may precipitate methylated CHH and CHG trinucleotide containing DNA fragments in addition to DNA sequences with methylated CpG sites. DNA methylation functions as the signal for DNA-interacting proteins to maintain genome structure or regulate gene expression. Therefore, methyl-CpG binding domain could specifically enrich differentially methylated regions (DMRs) of physiological relevance [53].
The detailed analysis to compare MeDIP and MBD capture revealed that both enrichment techniques are sensitive enough to identify DMRs in human cancer cells. Both the single molecule real time sequencing technology (SMRT) and the single-molecule nanopore DNA sequencing platform could discriminate 5-methylcytosines from other DNA bases including 5-hydroxymethylcytosines even methyladenine independent of bisulfite conversion [54-55].
With many advantages such as less bias during template preparation, lower cost and better accuracy, such new techniques could offer more methods to detect DNA methylation with high specificity and sensitivity in addition to more potential DNA methylation based biomarkers for cancer detection and screening.
Potential DNA methylation biomarkers for cancer detectionIt has been questioned whether the methylated DNA in the circulation is sensitive to detect cancers early enough for curative resection. However, the development of sensitive detection methods confirmed the potential value of DNA methylation in cancer detection (Table 1).
Most of DNA methylation biomarkers are well-known tumor suppressor genes silenced in primary tumor tissues. For example, currently well-used biomarkers such as AFP (Alpha-Fetal Protein), PSA (Prostate-specific antigen) and CEA (Carcinoembryonic antigen) are not tumor suppressor genes with important biological functions. Profiling of methylated DNA in the circulation instead of primary tumor tissues with MeDIP or MBD capture or other methylation specific analyses methods would identify more potential biomarks rather than functional important tumor suppressor genes.

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