The recently suggested intestinal cancer stem cell marker DCLK1 is an epigenetic biomarker for colorectal cancer

Recently, Dclk1 expression was identified to be an intestinal cancer stem cell specific biomarker in mouse models, implicating a potential role for targeting the DCLK1-postive cancer cells as a treatment for colorectal cancer. Using quantitative methylation specific PCR (qMSP) we here demonstrated that the DCLK1 promoter is hypermethylated in the vast majority of colorectal cancers (134/164; 82%), with no methylation in the normal mucosa samples (0/106). We further showed by Affymetrix exon arrays that DCLK1 is significantly downregulated in human colorectal cancer (n = 125) compared with normal colonic mucosa (n = 15), which was further confirmed by real-time RT-PCR of a subgroup of the samples. Additionally, a significant negative correlation was observed between methylation and DCLK1 expression in 74 cancer cell lines derived from 15 different tissues, and gene expression increased significantly after epigenetic drug treatment of initially methylated cancer cell lines. These findings underscore the potential of DCLK1 as a colorectal cancer biomarker for early detection, but may also have clinical implications regarding the previously proposed therapy toward DCLK1-positive cancer cells. This therapy would at best affect the cancer stem cell population, but will, based on the present results, not be efficient to treat the bulk of the tumor.     

p16INK4a and p14ARF methylation as a potential biomarker for human bladder cancer

Promoter hypermethylation is one of the putative mechanisms underlying the inactivation of negative cell-cycle regulators. We examined whether the methylation status of p16(INK4a) and p14(ARF), genes located upstream of the RB and p53 pathway, is a useful biomarker for the staging, clinical outcome, and prognosis of human bladder cancer. Using methylation-specific PCR (MSP), we examined the methylation status of p16(INK4a) and p14(ARF) in 64 samples from 45 bladder cancer patients (34 males, 11 females). In 19 patients with recurrent bladder cancer, we examined paired tissue samples from their primary and recurrent tumors. The methylation status of representative samples was confirmed by bisulfite DNA sequencing analysis. The median follow-up duration was 34.3 months (range 27.0-100.1 months). The methylation rate for p16(INK4a) and p14(ARF) was 17.8% and 31.1%, respectively, in the 45 patients. The incidence of p16(INKa) and p14(ARF) methylation was significantly higher in patients with invasive (>or=pT2) than superficial bladder cancer (pT1) (p=0.006 and p=0.001, respectively). No MSP bands for p16(INK4a) and p14(ARF) were detected in the 8 patients with superficial, non-recurrent tumors. In 19 patients with tumor recurrence, the p16(INK4a) and p14(ARF) methylation status of the primary and recurrent tumors was similar. Of the 22 patients who had undergone cystectomy, 8 (36.4%) manifested p16(INKa) methylation; p16(INK4a) was not methylated in 23 patients without cystectomy (p=0.002). Kaplan-Meier analysis revealed that patients with p14(ARF) methylation had a significantly poorer prognosis than those without (p=0.029). This is the first study indicating that MSP analysis of p16(INK4a) and p14(ARF) genes is a useful biomarker for the pathological stage, clinical outcome, and prognosis of patients with bladder cancer.     

Reduced activity of Arabidopsis chromosome-cohesion regulator gene CTF7/ECO1 alters cytosine methylation status and retrotransposon expression

Multicellular organisms such as higher plants require timely regulation of DNA replication and cell division to grow and develop. Recent work in Arabidopsis has shown that chromosome segregation during meiosis and mitosis depends on the activity of several genes that in yeast are involved in the establishment of chromosomal cohesion. In this process, proteins of the STRUCTURAL MAINTENANCE OF CHROMOSOMES (SMC) family tether chromosomes and establish inter- and intrachromosomal connections. In Arabidopsis, recruitment of SMC proteins and establishment of cohesion during key stages of the cell cycle depend on the activity of CHROMOSOME TRANSMISSION FIDELITY 7/ESTABLISHMENT OF COHESION 1 (CTF7/ECO1). Here we show that loss of CTF7/ECO1 activity alters the status of cytosine methylation in both intergenic regions and transposon loci. An increase in expression was also observed for transposon copia28, which suggests a link between CTF7/ECO1 activity, DNA methylation and gene silencing. More work is needed to determine the mechanistic relationships that intervene in this process.     

Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma

Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pN+ status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.     

Novel markers of gene methylation and expression in breast cancer

An optimized methylation-sensitive restriction fingerprinting technique was used to search for differentially methylated CpG islands in the tumor genome and detected seven genes subject to abnormal epigenetic regulation in breast cancer: SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, CACNG4, and PSMF1. For each gene, the rate of promoter methylation and changes in expression were estimated in tumor and morphologically intact paired specimens of breast tissue (N = 100). Significant methylation rates of 38, 18, and 8% were found for SEMA6B, BIN1, and LAMC3, respectively. The genes were not methylated in morphologically intact breast tissue. The expression of SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, CACNG4, and PSMF1 was decreased in 44–94% of tumor specimens by the real-time RT-PCR assay. The most profound changes in SEMA6B and LAMC3 suggest that these genes can be included in biomarker panels for breast cancer diagnosis. Fine methylation mapping of the most frequently methylated CpG islands (SEMA6B, BIN1, and LAMC3) provides a fundamental basis for developing efficient methylation tests for these genes.     

HOTAIRM1 as a potential biomarker for diagnosis of colorectal cancer functions the role in the tumour suppressor

Recent studies have revealed many different long noncoding RNAs (lncRNA), however, the investigation for their function and clinical value as tumour biomarkers has scarcely begun. Here, we found that expression of HOTAIRM1 was reduced in colorectal cancer (CRC) tissues compared with matched normal tissues, and plasma HOTAIRM1 levels in CRC patients were less than in controls. The cut‐off point was chosen as 0.003 with a sensitivity of 64.00% and a specificity of 76.50% in the validation set. The performance of HOTAIRM1 was highly comparable to carcinoembryonic antigen (CEA), and better than CA19‐9 and CA125. The combined assay of HOTAIRM1 and CEA raised the sensitivity and specificity to 84.00%. HOTAIRM1 knockdown resulted in obvious changes in expression of the cell proliferation related to genes and promoted cell proliferation. HOTAIRM1 plays a role of tumour suppressor in CRC; Down‐regulation of HOTAIRM1 can serve as a biomarker for CRC, and combined HOTAIRM1 and CEA assay might provide a promising diagnosis for CRC.     

乳腺癌相关基因的甲基化

肿瘤组织中常伴随基因组整体甲基化水平降低和(或)某些基因CpG岛甲基化水平异常升高,这两种变化在肿瘤发生和发展中都扮演着重要的角色。近年来诸多研究报道了CpG岛高甲基化可导致乳腺癌相关的一系列关键基因的表达缺失。由于表观遗传变化存在潜在可逆性,因此,通过检测患者特定基因甲基化状态早期诊断乳腺癌,以及运用甲基化抑制剂来治疗乳腺癌,已成为国内外研究的热点和新思路。     

The MTHFR C677T polymorphism and global DNA methylation in oral epithelial cells

DNA methylation is mediated by DNA methyltransferases (DNMTs) that add a methyl group to the 5′-carbon of cytosine. The enzyme methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate in the rate-limiting step of the cycle involving the methyl donor S-adenosyl-L-methionine (SAM). The MTHFR C677T polymorphism results in a thermolabile enzyme with reduced activity that is predicted to influence the DNA methylation status. In this study, we investigated the impact of the MTHFR C677T polymorphism on the global DNA methylation of oral epithelial cells obtained from 54 healthy subjects. There were no significant differences in global DNA methylation among the MTHFR CC, CT and TT genotypes (p = 0.75; Kruskal-Wallis test).     

Comprehensive serum N-glycan profiling identifies a biomarker panel for early diagnosis of non-small-cell lung cancer

Aberrant serum N-glycan profiles have been observed in multiple cancers including non-small-cell lung cancer (NSCLC), yet the potential of N-glycans in the early diagnosis of NSCLC remains to be determined. In this study, serum N-glycan profiles of 275 NSCLC patients and 309 healthy controls were characterized by MALDI-TOF-MS. The levels of serum N-glycans and N-glycosylation patterns were compared between NSCLC and control groups. In addition, a panel of N-glycan biomarkers for NSCLC diagnosis was established and validated using machine learning algorithms. As a result, a total of 54 N-glycan structures were identified in human serum. Compared with healthy controls, 29 serum N-glycans were increased or decreased in NSCLC patients. N-glycan abundance in different histological types or clinical stages of NSCLC presented differentiated changes. Furthermore, an optimal biomarker panel of eight N-glycans was constructed based on logistic regression, with an AUC of 0.86 in the validation set. Notably, this model also showed a desirable capacity in distinguishing early-stage patients from healthy controls (AUC = 0.88). In conclusion, our work highlights the abnormal N-glycan profiles in NSCLC and provides supports potential application of N-glycan biomarker panel in clinical NSCLC detection.     

Identification of CD28 and PTEN as novel prognostic markers for cervical cancer

Cervical cancer (CC) is the most common malignant tumor with poor clinical outcome among women. Identification of novel biomarkers could be beneficial for the clinical diagnosis and treatment of CC. This study aimed to identify prognostic biomarkers for the prediction of prognostic status of CC patients, and explore the effect of the corresponding methylated genes in the occurrence and development of CC. The methylation microarray data of CC was extracted from The Cancer Genome Atlas (TCGA) dataset. The methylation genes associated with the prognostic status were identified based on the information of the relapse-free survival (RFS) of the CC patients. The prognostic gene pairs were further identified. Then, the prognostic signature was identified by the forward search algorithm based on the C-index method. The results were validated by independent dataset. Finally, the functional analysis was performed on the methylation genes. A total of 276 methylation genes and 2508 gene pairs associated with the prognostic status of the CC were identified. A signature composed of eight methylation gene pairs was obtained to predict the prognostic status of cervical patients. A series of genes that played an important role in the occurrence and development of CC were obtained by the functional enrichment analysis. To summary, a prognostic signature consisting of eight methylation gene pairs was obtained. Of note, the CD28 and PTEN gene pair were found to play important roles in the occurrence and development of CC.     

Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.     

The recently suggested intestinal cancer stem cell marker DCLK1 is an epigenetic biomarker for colorectal cancer

Recently, Dclk1 expression was identified to be an intestinal cancer stem cell specific biomarker in mouse models, implicating a potential role for targeting the DCLK1-postive cancer cells as a treatment for colorectal cancer. Using quantitative methylation specific PCR (qMSP) we here demonstrated that the DCLK1 promoter is hypermethylated in the vast majority of colorectal cancers (134/164; 82%), with no methylation in the normal mucosa samples (0/106). We further showed by Affymetrix exon arrays that DCLK1 is significantly downregulated in human colorectal cancer (n = 125) compared with normal colonic mucosa (n = 15), which was further confirmed by real-time RT-PCR of a subgroup of the samples. Additionally, a significant negative correlation was observed between methylation and DCLK1 expression in 74 cancer cell lines derived from 15 different tissues, and gene expression increased significantly after epigenetic drug treatment of initially methylated cancer cell lines. These findings underscore the potential of DCLK1 as a colorectal cancer biomarker for early detection, but may also have clinical implications regarding the previously proposed therapy toward DCLK1-positive cancer cells. This therapy would at best affect the cancer stem cell population, but will, based on the present results, not be efficient to treat the bulk of the tumor.     

Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.     

A systematic comparison of quantitative high-resolution DNA methylation analysis and methylation-specific PCR

Assessment of DNA methylation has become a critical factor for the identification, development and application of methylation based biomarkers. Here we describe a systematic comparison of a quantitative high-resolution mass spectrometry-based approach (MassARRAY), pyrosequencing and the broadly used methylation-specific PCR (MSP) technique analyzing clinically relevant epigenetically silenced genes in acute myeloid leukemia (AML). By MassARRAY and pyrosequencing, we identified significant DNA methylation differences at the ID4 gene promoter and in the 5′ region of members of the SFRP gene family in 62 AML patients compared with healthy controls. We found a good correlation between data obtained by MassARRAY and pyrosequencing (correlation coefficient R² = 0.88). MSP-based assessment of the identical samples showed less pronounced differences between AML patients and controls. By direct comparison of MSP-derived and MassARRAY-based methylation data as well as pyrosequencing, we could determine overestimation of DNA methylation data by MSP. We found sequence-context dependent highly variable cut-off values of quantitative DNA methylation values serving as discriminator for the two MSP methylation categories. Moreover, good agreements between quantitative methods and MSP could not be achieved for all investigated loci. Significant correlation of the quantitative assessment but not of MSP-derived methylation data with clinically important characteristics in our patient cohort demonstrated clinical relevance of quantitative DNA methylation assessment. Taken together, while MSP is still the most commonly applied technique for DNA methylation assessment, our data highlight advantages of quantitative approaches for precise characterization and reliable biomarker use of aberrant DNA methylation in primary patient samples, particularly.