DNA methylation regulates Microtubule-associated tumor suppressor 1 in human non-small cell lung carcinoma

Microtubule associated tumor suppressor 1 (MTUS1) has been recognized as a tumor suppressor gene in multiple cancers. However, the molecular mechanisms underlying the regulation of MTUS1 are yet to be investigated. This study aimed to clarify the significance of DNA methylation in silencing MTUS1 expression. We report that MTUS1 acts as tumor suppressor in non-small cell lung carcinoma (NSCLC). Analysis of in silico database and subsequent knockdown of DNMT1 suggested an inverse correlation between DNMT1 and MTUS1 function. Interestingly, increased methylation at MTUS1 promoter is associated with low expression of MTUS1. Treatment with DNA methyltransferases (DNMTs) inhibitor, 5-aza-2′-deoxycytidine (AZA) leads to both reduced promoter methylation accompanied with enrichment of H3K9Ac and enhanced MTUS1 expression. Remarkably, knockdown of MTUS1 showed increased proliferation and migration of NSCLC cells in contrast to diminished proliferation and migration, upon treatment with AZA. We concluded that low expression of MTUS1 correlates to DNA methylation and histone deacetylation in human NSCLC.     

DNA methylation and breast tumor clinicopathological features: The Western New York Exposures and Breast Cancer (WEB) study

We evaluated the association between methylation of 9 genes, SCGB3A1, GSTP1, RARB, SYK, FHIT, CDKN2A, CCND2, BRCA1, and SFN in tumor samples from 720 breast cancer cases with clinicopathological features of the tumors and survival. Logistic regression was used to estimate odds ratios (OR) of methylation and Cox proportional hazards models to estimate hazard ratios (HR) between methylation and breast cancer related mortality. Estrogen receptor (ER) and progesterone receptor (PR) positivity were associated with increased SCGB3A1 methylation among pre- and post-menopausal cases. Among premenopausal women, compared with Stage 0 cases, cases of invasive cancer were more likely to have increased methylation of RARB (Stage I OR = 4.7, 95% CI: 1.1–19.0; Stage IIA/IIB OR = 9.7, 95% CI: 2.4–39.9; Stage III/IV OR = 5.6, 95% CI: 1.1–29.4) and lower methylation of FHIT (Stage I OR = 0.2, 95% CI: 0.1–0.9; Stage IIA/IIB OR = 0.2, 95% CI: 0.1–0.8; Stage III/IV OR = 0.6, 95% CI: 0.1–3.4). Among postmenopausal women, methylation of SYK was associated with increased tumor size (OR = 1.7, 95% CI: 1.0–2.7) and higher nuclear grade (OR = 2.0, 95% CI 1.2–3.6). Associations between methylation and breast cancer related mortality were observed among pre- but not post-menopausal women. Methylation of SCGB3A1 was associated with reduced risk of death from breast cancer (HR = 0.41, 95% CI: 0.17–0.99) as was BRCA1 (HR = 0.41, 95% CI: 0.16–0.97). CCND2 methylation was associated with increased risk of breast cancer mortality (HR = 3.4, 95% CI: 1.1–10.5). We observed differences in methylation associated with tumor characteristics; methylation of these genes was also associated with breast cancer survival among premenopausal cases. Understanding of the associations of DNA methylation with other clinicopathological features may have implications for prevention and treatment.     

The underlying pathophysiology association between the Type 2-diabetic and hepatocellular carcinoma

Type 2-diabetic (T2D) disease has been reported to increase the incidence of liver cancer, however, the underlying pathophysiology is still not fully understood. Here, we aimed to reveal the underlying pathophysiology association between the T2D and hepatocellular carcinoma (HCC) and, therefore, to find the possible therapeutic targets in the occurrence and development of HCC. The methylation microarray data of T2D and HCC were extracted from the Gene Expression Omnibus and The Cancer Genome Atlas. A total of 504 differentially methylated genes (DMGs) between T2D samples and the controls were identified, whereas 6269 DMGs were identified between HCC samples and the control groups. There were 336 DMGs coexisting in diabetes and HCC, among which 86 genes were comethylated genes. These genes were mostly enriched in pathways as glycosaminoglycan biosynthesis, fatty acid, and metabolic pathway as glycosaminoglycan degradation and thiamine, fructose and mannose. There were 250 DMGs that had differential methylation direction between T2D DMGs and HCC DMGs, and these genes were enriched in the Sphingolipid metabolism pathway and immune pathways through natural killer cell-mediated cytotoxicity and ak-STAT signaling pathway. Eight genes were found related to the occurrence and development of diabetes and HCC. Moreover, the result of protein-protein interaction network showed that CDKN1A gene was related to the prognosis of HCC. In summary, eight genes were found to be associated with the development of HCC and CDKN1A may serve as the potential prognostic gene for HCC.     

Comparison of bisulfite sequencing PCR with pyrosequencing for measuring differences in DNA methylation

DNA methylation strongly affects chromatin structure and the regulation of gene expression. For many years, bisulfite sequencing PCR (BSP) has served as the “gold standard” for measuring DNA methylation. However, with the evolution of pyrosequencing as a tool to evaluate DNA methylation, the need arises to compare the relative efficiencies of the two techniques in measuring DNA methylation. We provide for the first time a direct assessment of BSP and pyrosequencing to detect and quantify hypomethylation, hypermethylation, and mixed methylation of the ABCB1 promoter in various drug-sensitive and drug-resistant MCF-7 breast cancer cell lines through head-to-head experimentation. Our findings indicate that although both methods can reliably detect increased, decreased, and mixed methylation of DNA, BSP appears to be more sensitive than pyrosequencing at detecting strong hypermethylation of DNA. However, we also observed greater variability in the methylation of CpG sites by BSP, possibly due to the additional bacterial cloning step required by BSP over pyrosequencing. BSP and pyrosequencing equally detected hypomethylation and mixed methylation of DNA. The ability of pyrosequencing to reliably detect differences in DNA methylation across cell populations without requiring the cloning of bisulfite-treated DNA into bacterial expression vectors was seen as a major advantage of this technique.     

Promoter hypermethylation of tumor suppressor genes in serum as potential biomarker for the diagnosis of nasopharyngeal carcinoma

Purpose: Promoter hypermethylation of tumor suppressor genes may serve as a promising biomarker for the diagnosis of cancer. Cell-free circulating DNA (cf-DNA) shares hypermethylation status with primary tumors. This study investigated promoter hypermethylation of five tumor suppressor genes as markers in the detection of nasopharyngeal carcinoma (NPC) in serum samples. Methods: cf-DNA was extracted from serum collected from 40 NPC patients and 41 age- and sex-matched healthy subjects. The promoter hypermethylation status of the five genes (RASSF1, CDKN2A, DLEC1, DAPK1 and UCHL1) was assessed by methylation-specific PCR after sodium bisulfite conversion. Differences in the methylation status of these five genes between NPC patients and healthy subjects were compared. Results: The concentration of cf-DNA in the serum of NPC patients was significantly higher than that in normal controls. The five tumor suppressor genes – RASSF1, CDKN2A, DLEC1, DAPK1 and UCHL1 – were found to be methylated in 17.5%, 22.5%, 25.0%, 51.4% and 64.9% of patients, respectively. The combination of four-gene marker – CDKN2A, DLEC1, DAPK1 and UCHL1 – had the highest sensitivity and specificity in predicting NPC. Conclusion: Screening DNA hypermethylation of tumor suppressor genes in serum was a promising approach for the diagnosis of NPC.     

Efficacy and epigenetic interactions of novel DNA hypomethylating agent guadecitabine (SGI-110) in preclinical models of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a deadly malignancy characterized at the epigenetic level by global DNA hypomethylation and focal hypermethylation on the promoter of tumor suppressor genes. In most cases it develops on a background of liver steatohepatitis, fibrosis, and cirrhosis. Guadecitabine (SGI-110) is a second-generation hypomethylating agent, which inhibits DNA methyltransferases. Guadecitabine is formulated as a dinucleotide of decitabine and deoxyguanosine that is resistant to cytidine deaminase (CDA) degradation and results in prolonged in vivo exposure to decitabine following small volume subcutaneous administration of guadecitabine. Here we found that guadecitabine is an effective demethylating agent and is able to prevent HCC progression in pre-clinical models. In a xenograft HCC HepG2 model, guadecitabine impeded tumor growth and inhibited angiogenesis, while it could not prevent liver fibrosis and inflammation in a mouse model of steatohepatitis. Demethylating efficacy of guadecitabine on LINE-1 elements was found to be the highest 8 d post-infusion in blood samples of mice. Analysis of a panel of human HCC vs. normal tissue revealed a signature of hypermethylated tumor suppressor genes (CDKN1A, CDKN2A, DLEC1, E2F1, GSTP1, OPCML, E2F1, RASSF1, RUNX3, and SOCS1) as detected by methylation-specific PCR. A pronounced demethylating effect of guadecitabine was obtained also in the promoters of a subset of tumor suppressors genes (CDKN2A, DLEC1, and RUNX3) in HepG2 and Huh-7 HCC cells. Finally, we analyzed the role of macroH2A1, a variant of histone H2A, an oncogene upregulated in human cirrhosis/HCC that synergizes with DNA methylation in suppressing tumor suppressor genes, and it prevents the inhibition of cell growth triggered by decitabine in HCC cells. Guadecitabine, in contrast to decitabine, blocked growth in HCC cells overexpressing macroH2A1 histones and with high CDA levels, despite being unable to fully demethylate CDKN2A, RUNX3, and DLEC1 promoters altered by macroH2A1. Collectively, our findings in human and mice models reveal novel epigenetic anti-HCC effects of guadecitabine, which might be effective specifically in advanced states of the disease.     

Hypermethylated RASSF1 and SLC5A8 promoters alongside BRAFV600E mutation as biomarkers for papillary thyroid carcinoma

Circulating cell-free DNA (cfDNA) has been considered as a diagnostic source to track genetic and epigenetic alterations in cancer. We aimed to study mutation in addition to the methylation status in the promoter regions of RASSF1 and SLC5A8 genes in tissues and circulating free DNA samples of patients affected with papillary thyroid carcinoma (PTC) and thyroid nodules as controls. BRAF^V600E mutation was studied by ARMS-scorpion real-time polymerase chain reaction method in 57 PTC and 45 thyroid nodule cases. Methylation status of RASSF1 and SLC5A8 promoter regions was analyzed by methylation-specific high-resolution melting curve analysis. BRAF^V600E mutation was found in 39 (68.4%) out of 57 PTC tissue samples, while in 33 (49.1%) cases of cfDNA, this mutation was detected. The frequency of BRAF^V600E mutation in cfDNA was significantly different between metastatic and nonmetastatic PTC cases (22 of 33 PTC cases vs. 5 of 34 thyroid nodule samples). Methylation levels of three promoter regions of SLC5A8 and proximal promoter region of RASSF1 was significantly different between PTC and thyroid nodule cases in both cfDNA and tissue DNA. In addition, the methylation status of these two genes in tissue DNA was reflected in methylation status observed in cfDNA. This study confirmed that BRAF^V600E mutation is better for discrimination between papillary thyroid carcinoma and thyroid nodules. On the other hand, hypermethylation in the more proximal promoter regions to RASSF1 and SLC5A8 genes showed higher sensitivity and more acceptable specificity for this discrimination.     

Detection and monitoring of HBV-related hepatocellular carcinoma from plasma cfDNA fragmentation profiles

Most hepatocellular carcinomas (HCCs) are associated with hepatitis B virus infection (HBV) in China. Early detection of HCC can significantly improve prognosis but is not yet fully clinically feasible. This study aims to develop methods for detecting HCC and studying the carcinogenesis of HBV using plasma cell-free DNA (cfDNA) whole-genome sequencing (WGS) data. Low coverage WGS was performed for 452 participants, including healthy individuals, hepatitis B patients, cirrhosis patients, and HCC patients. Then the sequencing data were processed using various machine learning models based on cfDNA fragmentation profiles for cancer detection. Our best model achieved a sensitivity of 87.10% and a specificity of 88.37%, and it showed an increased sensitivity with higher BCLC stages of HCC. Overall, this study proves the potential of a non-invasive assay based on cfDNA fragmentation profiles for the detection and prognosis of HCC and provides preliminary data on the carcinogenic mechanism of HBV.     

Insights into the role of components of the tumor microenvironment in oral carcinoma call for new therapeutic approaches

The research on oral cancer has focused mainly on the cancer cells, their genetic changes and consequent phenotypic modifications. However, it is increasingly clear that the tumor microenvironment (TME) has been shown to be in a dynamic state of inter-relations with the cancer cells. The TME contains a variety of components including the non-cancerous cells (i.e., immune cells, resident fibroblasts and angiogenic vascular cells) and the ECM milieu [including fibers (mainly collagen and fibronectin) and soluble factors (i.e., enzymes, growth factors, cytokines and chemokines)]. Thus, it is currently assumed that TME is considered a part of the cancerous tissue and the functionality of its key components constitutes the setting on which the hallmarks of the cancer cells can evolve. Therefore, in terms of controlling a malignancy, one should control the growth, invasion and spread of the cancer cells through modifications in the TME components. This mini review focuses on the TME as a diagnostic approach and reports the recent insights into the role of different TME key components [such as carcinoma-associated fibroblasts (CAFs) and inflammation (CAI) cells, angiogenesis, stromal matrix molecules and proteases] in the molecular biology of oral carcinoma. Furthermore, the impact of TME components on clinical outcomes and the concomitant need for development of new therapeutic approaches will be discussed.     

Immune checkpoint blockade and its combination therapy with small-molecule inhibitors for cancer treatment

Initially understood for its physiological maintenance of self-tolerance, the immune checkpoint molecule has recently been recognized as a promising anti-cancer target. There has been considerable interest in the biology and the action mechanism of the immune checkpoint therapy, and their incorporation with other therapeutic regimens. Recently the small-molecule inhibitor (SMI) has been identified as an attractive combination partner for immune checkpoint inhibitors (ICIs) and is becoming a novel direction for the field of combination drug design. In this review, we provide a systematic discussion of the biology and function of major immune checkpoint molecules, and their interactions with corresponding targeting agents. With both preclinical studies and clinical trials, we especially highlight the ICI + SMI combination, with its recent advances as well as its application challenges.     

Vascular-homing peptides for targeted drug delivery and molecular imaging: Meeting the clinical challenges

The vasculature of each organ expresses distinct molecular signatures critically influenced by the pathological status. The heterogeneous profile of the vascular beds has been successfully unveiled by the in vivo phage display, a high-throughput tool for mapping normal, diseased, and tumor vasculature. Specific challenges of this growing field are targeted therapies against cancer and cardiovascular diseases, as well as novel bioimaging diagnostic tools. Tumor vasculature-homing peptides have been extensively evaluated in several preclinical and clinical studies both as targeted-therapy and diagnosis. To date, results from several Phase I and II trials have been reported and many other trials are currently ongoing or recruiting patients. In this review, advances in the identification of novel peptide ligands and their corresponding receptors on tumor endothelium through the in vivo phage display technology are discussed. Emphasis is given to recent findings in the clinical setting of vascular-homing peptides selected by in vivo phage display for the treatment of advanced malignancies and their altered vascular beds.     

Chromosomal microarray analysis of consecutive individuals with autism spectrum disorders or learning disability presenting for genetic services

Chromosomal microarray analysis is now commonly used in clinical practice to identify copy number variants (CNVs) in the human genome. We report our experience with the use of the 105 K and 180 K oligonucleotide microarrays in 215 consecutive patients referred with either autism or autism spectrum disorders (ASD) or developmental delay/learning disability for genetic services at the University of Kansas Medical Center during the past 4 years (2009–2012). Of the 215 patients [140 males and 75 females (male/female ratio = 1.87); 65 with ASD and 150 with learning disability], abnormal microarray results were seen in 45 individuals (21%) with a total of 49 CNVs. Of these findings, 32 represented a known diagnostic CNV contributing to the clinical presentation and 17 represented non-diagnostic CNVs (variants of unknown significance). Thirteen patients with ASD had a total of 14 CNVs, 6 CNVs recognized as diagnostic and 8 as non-diagnostic. The most common chromosome involved in the ASD group was chromosome 15. For those with a learning disability, 32 patients had a total of 35 CNVs. Twenty-six of the 35 CNVs were classified as a known diagnostic CNV, usually a deletion (n = 20). Nine CNVs were classified as an unknown non-diagnostic CNV, usually a duplication (n = 8). For the learning disability subgroup, chromosomes 2 and 22 were most involved. Thirteen out of 65 patients (20%) with ASD had a CNV compared with 32 out of 150 patients (21%) with a learning disability. The frequency of chromosomal microarray abnormalities compared by subject group or gender was not statistically different. A higher percentage of individuals with a learning disability had clinical findings of seizures, dysmorphic features and microcephaly, but not statistically significant. While both groups contained more males than females, a significantly higher percentage of males were present in the ASD group.