5-Hydroxymethylome in Circulating Cell-free DNA as A Potential Biomarker for Non-small-cell Lung Cancer

Non-small-cell lung cancer(NSCLC), the most common type of lung cancer accounting for 85% of the cases, is often diagnosed at advanced stages owing to the lack of efficient early diagnostic tools. 5-Hydroxymethylcytosine(5 hmC) signatures in circulating cell-free DNA(cfDNA) that carries the cancer-specific epigenetic patterns may represent the valuable biomarkers for discriminating tumor and healthy individuals, and thus could be potentially useful for NSCLC diagnosis. Here,we employed a sensitive and reliable method to map genome-wide 5 hmC in the cfDNA of Chinese NSCLC patients and detected a significant 5 hmC gain in both the gene bodies and promoter regions in the blood samples from tumor patients compared with healthy controls. Specifically, we identified six potential biomarkers from 66 patients and 67 healthy controls(mean decrease accuracy3.2, P 3.68E-19) using machine-learning-based tumor classifiers with high accuracy. Thus,the unique signature of 5 hmC in tumor patient's cfDNA identified in our study may provide valuable information in facilitating the development of new diagnostic and therapeutic modalities for NSCLC.     

Integrated fragmentomic profile and 5-Hydroxymethylcytosine of capture-based low-pass sequencing data enables pan-cancer detection via cfDNA

BackgroundUsing epigenetic markers and fragmentomics of cell-free DNA for cancer detection has been proven applicable.MethodsWe further investigated the diagnostic potential of combining two features (epigenetic markers and fragmentomic information) of cell-free DNA for detecting various types of cancers. To do this, we extracted cfDNA fragmentomic features from 191 whole-genome sequencing data and studied them in 396 low-pass 5hmC sequencing data, which included four common cancer types and control samples.ResultsIn our analysis of 5hmC sequencing data from cancer samples, we observed aberrant ultra-long fragments (220–500 bp) that differed from normal samples in terms of both size and coverage profile. These fragments played a significant role in predicting cancer. Leveraging the ability to detect cfDNA hydroxymethylation and fragmentomic markers simultaneously in low-pass 5hmC sequencing data, we developed an integrated model that incorporated 63 features representing both fragmentomic features and hydroxymethylation signatures. This model achieved high sensitivity and specificity for pan-cancer detection (88.52% and 82.35%, respectively).ConclusionWe showed that fragmentomic information in 5hmC sequencing data is an ideal marker for cancer detection and that it shows high performance in low-pass sequencing data.     

SARI suppresses colitis‐associated cancer development by maintaining MCP‐1‐mediated tumour‐associated macrophage recruitment

SARI (suppressor of AP‐1, regulated by IFN) impaired tumour growth by promoting apoptosis and inhibiting cell proliferation and tumour angiogenesis in various cancers. However, the role of SARI in regulating tumour‐associated inflammation microenvironment is still elusive. In our study, the colitis‐dependent and ‐independent primary model were established in SARI deficiency mice and immuno‐reconstructive mice to investigate the functional role of SARI in regulating tumour‐associated inflammation microenvironment and primary colon cancer formation. The results have shown that SARI deficiency promotes colitis‐associated cancer (CAC) development only in the presence of colon inflammation. SARI inhibited tumour‐associated macrophages (TAM) infiltration in colon tissues, and SARI deficiency in bone marrow cells has no observed role in the promotion of intestinal tumorigenesis. Mechanism investigations indicated that SARI down‐regulates p‐STAT1 and STAT1 expression in colon cancer cells, following inhibition of MCP‐1/CCR2 axis activation during CAC development. Inverse correlations between SARI expression and macrophage infiltration, MCP‐1 expression and p‐STAT1 expression were also demonstrated in colon malignant tissues. Collectively, our results prove the inhibition role of SARI in colon cancer formation through regulating TAM infiltration.     

Mutational landscape and genetic signatures of cell‐free DNA in tumour‐induced osteomalacia

Tumour‐induced osteomalacia (TIO) is a very rare paraneoplastic syndrome with bone pain, fractures and muscle weakness, which is mostly caused by phosphaturic mesenchymal tumours (PMTs). Cell‐free DNA (cfDNA) has been regarded as a non‐invasive liquid biopsy for many malignant tumours. However, it has not been studied in benign tumours, which prompted us to adopt the targeted next‐generation sequencing approach to compare cfDNAs of 4 TIO patients, four patients with bone metastasis (BM) and 10 healthy controls. The mutational landscapes of cfDNA in TIO and BM groups were similar in the spectrum of allele frequencies and mutation types. Markedly, deleterious missense mutations in FGFR1 and loss‐of‐function mutations in MED12 were found in 3/4 TIO patients but none of BM patients. The gene ontology analysis strongly supported that these mutated genes found in TIOs would play a potential role in PMTs' process. The genetic signatures and corresponding change in expression of FGFR1 and FGF23 were further validated in PMT tissues from a test cohort of another three TIO patients. In summary, we reported the first study of the mutational landscape and genetic signatures of cfDNA in TIO/PMTs.     

TET3‐mediated DNA oxidation promotes ATR‐dependent DNA damage response

An efficient, accurate, and timely DNA damage response (DDR) is crucial for the maintenance of genome integrity. Here, we report that ten‐eleven translocation dioxygenase (TET) 3‐mediated conversion of 5‐methylcytosine (5mC) to 5‐hydroxymethylcytosine (5hmC) in response to ATR‐dependent DDR regulates DNA repair. ATR‐dependent DDR leads to dynamic changes in 5hmC levels and TET3 enzymatic activity. We show that TET3 is an ATR kinase target that oxidizes DNA during ATR‐dependent DNA damage repair. Modulation of TET3 expression and activity affects DNA damage signaling and DNA repair and consequently cell death. Our results provide novel insight into ATR‐mediated DDR, in which TET3‐mediated DNA demethylation is crucial for efficient DNA repair and maintenance of genome stability.     

The diverse origins of circulating cell‐free DNA in the human body: a critical re‐evaluation of the literature

Since the detection of cell‐free DNA (cfDNA) in human plasma in 1948, it has been investigated as a non‐invasive screening tool for many diseases, especially solid tumours and foetal genetic abnormalities. However, to date our lack of knowledge regarding the origin and purpose of cfDNA in a physiological environment has limited its use to more obvious diagnostics, neglecting, for example, its potential utility in the identification of predisposition to disease, earlier detection of cancers, and lifestyle‐induced epigenetic changes. Moreover, the concept or mechanism of cfDNA could also have potential therapeutic uses such as in immuno‐ or gene therapy. This review presents an extensive compilation of the putative origins of cfDNA and then contrasts the contributions of cellular breakdown processes with active mechanisms for the release of cfDNA into the extracellular environment. The involvement of cfDNA derived from both cellular breakdown and active release in lateral information transfer is also discussed. We hope to encourage researchers to adopt a more holistic view of cfDNA research, taking into account all the biological pathways in which cfDNA is involved, and to give serious consideration to the integration of in vitro and in vivo research. We also wish to encourage researchers not to limit their focus to the apoptotic or necrotic fraction of cfDNA, but to investigate the intercellular messaging capabilities of the actively released fraction of cfDNA and to study the role of cfDNA in pathogenesis.     

CCL18 promotes the metastasis of squamous cell carcinoma of the head and neck through MTDH‐NF‐κB signalling pathway

Metastasis is one of the primary causes for high mortality in patients with squamous cell carcinoma of the head and neck (SCCHN). Our previous study showed that chemokine (C‐C motif) ligand 18 (CCL18), derived from tumour‐associated macrophages (TAMs), regulates SCCHN metastasis by promoting epithelial‐mesenchymal transition (EMT) and preserving stemness. However, the underlying mechanism needs to be further investigation. Interestingly, metadherin (MTDH) expression was induced when SCCHN cells were stimulated with recombinant CCL18 protein in this study. Suppressing MTDH expression reversed CCL18‐induced migration, invasion and EMT in SCCHN cells. Furthermore, the NF‐κB signalling pathway was involved in the MTDH knock‐down cells with CCL18 stimulation. We performed ELISA to evaluate the CCL18 levels in the serums of 132 treatment‐naive SCCHN patients, 25 patients with precancerous lesion and 32 healthy donors. Our results demonstrated that serum CCL18 levels were significantly higher in SCCHN patients than patients with precancerous lesion and healthy individuals. CCL18 levels were found to be significantly correlated with tumour classification, clinical stage, lymph node metastasis and histological grade in SCCHN patients. Thus, our findings suggest that CCL18 may serve as a potential biomarker for diagnosis of SCCHN and promote SCCHN invasion, migration and EMT by MTDH‐NF‐κB signalling pathway.     

Toll‐like receptor 4 regulates spontaneous intestinal tumorigenesis by up‐regulating IL‐6 and GM‐CSF

Inflammation is as an important component of intestinal tumorigenesis. The activation of Toll‐like receptor 4 (TLR4) signalling promotes inflammation in colitis of mice, but the role of TLR4 in intestinal tumorigenesis is not yet clear. About 80%–90% of colorectal tumours contain inactivating mutations in the adenomatous polyposis coli (Apc) tumour suppressor, and intestinal adenoma carcinogenesis in familial adenomatous polyposis (FAP) is also closely related to the germline mutations in Apc. The Apc^Min/+ (multiple intestinal neoplasia) model mouse is a well‐utilized model of FAP, an inherited form of intestinal cancer. In this study, Apc^Min/+ intestinal adenoma mice were generated on TLR4‐sufficient and TLR4‐deficient backgrounds to investigate the carcinogenic effect of TLR4 in mouse gut by comparing mice survival, peripheral blood cells, bone marrow haematopoietic precursor cells and numbers of polyps in the guts of Apc^Min/+ WT and Apc^Min/+ TLR4^?/? mice. The results revealed that TLR4 had a critical role in promoting spontaneous intestinal tumorigenesis. Significant differential genes were screened out by the high‐throughput RNA‐Seq method. After combining these results with KEGG enrichment data, it was determined that TLR4 might promote intestinal tumorigenesis by activating cytokine‐cytokine receptor interaction and pathways in cancer signalling pathways. After a series of validation experiments for the concerned genes, it was found that IL6, GM‐CSF (CSF2), IL11, CCL3, S100A8 and S100A9 were significantly decreased in gut tumours of Apc^Min/+ TLR4^?/? mice compared with Apc^Min/+ WT mice. In the functional study of core down‐regulation factors, it was found that IL6, GM‐CSF, IL11, CCL3 and S100A8/9 increased the viability of colon cancer cell lines and decreased the apoptosis rate of colon cancer cells with irradiation and chemical treatment.     

Circulating Free DNA as Biomarker and Source for Mutation Detection in Metastatic Colorectal Cancer

Background

Circulating cell-free DNA (cfDNA) in plasma has shown potential as biomarker in various cancers and could become an importance source for tumour mutation detection. The objectives of our study were to establish a normal range of cfDNA in a cohort of healthy individuals and to compare this with four cohorts of metastatic colorectal cancer (mCRC) patients. We also investigated the prognostic value of cfDNA and analysed the tumour-specific KRAS mutations in the plasma.

Methods

The study was a prospective biomarker evaluation in four consecutive Phase II trials, including 229 patients with chemotherapy refractory mCRC and 100 healthy individuals. Plasma was obtained from an EDTA blood-sample, and the total number of DNA alleles and KRAS mutated alleles were assessed using an in-house ARMS-qPCR as previously described.

Results

Median cfDNA levels were higher in mCRC compared to controls (p <0.0001). ROC analysis revealed an AUC of 0.9486 (p<0.00001). Data showed impaired OS with increasing levels of baseline cfDNA both when categorising patients by quartiles of cfDNA and into low or high cfDNA groups based on the upper normal range of the control group (Median OS 10.2 (8.3–11.7) and 5.2 (4.6–5.9) months, respectively, HR 1.78, p = 0.0006). Multivariate analysis confirmed an independent prognostic value of cfDNA (HR 1.5 (95% CI 1.3–1.7) for each increase in the cfDNA quartile). The overall concordance of KRAS mutations in plasma and tissue was high (85%).

Conclusions

These data confirm the prognostic value of cfDNA measurement in plasma and utility for mutation detection with the method presented.     

Transforming growth factor‐β1 modulates metalloproteinase‐2 and ‐9, nitric oxide,RhoA and α‐smooth muscle actin expression in colon adenocarcinoma cells

Colon carcinoma invasiveness is a process involving cell–cell and cell–matrix alterations, local proteolysis of the ECM (extracellular matrix) or changes in cytokine and growth factor levels. In order to evaluate the role of TGF‐β1 (transforming growth factor‐β1) and small G protein RhoA in tumour progression, the influence of TGF‐β1 treatment or RhoA‐associated kinase inhibitor on the production of NO (nitric oxide) and MMP‐2 and MMP‐9 (metalloproteinases‐2 and ‐9) was analysed in three human colon adenocarcinoma cell lines (HT29, LS180, SW948) representing different stages of tumour development. All the tested cell lines produced low amounts of MMP‐2 and MMP‐9. rhTGF‐β1 and the synthetic Rho kinase inhibitor (Y‐27632) decreased MMP‐2 secretion by colon cancer cells, especially in the most advanced stage of colon cancer. rhTGF‐β1 decreased NO secretion by cells, while Y‐27632 had no effect on it. Immunoblotting with anti‐RhoA antibodies followed by densitometry revealed that RhoA levels were slightly increased after incubation of colon carcinoma cells (SW948) with rhTGF‐β1. rhTGF‐β1 induced α‐smooth muscle actin (α‐SMA) expression, especially in high Duke's grade of colon cancer, while Y‐27632 blocked it. Summing up, in colon carcinoma cells, TGF‐β1 and RhoA protein may regulate tumour invasiveness measured as MMP, NO and α‐SMA expression or assayed using motility data and may be a good target for cancer therapy.     

5‐Hydroxymethylcytosine: a new kid on the epigenetic block?

The discovery of the Ten‐Eleven‐Translocation (TET) oxygenases that catalyze the hydroxylation of 5‐methylcytosine (5mC) to 5‐hydroxymethylcytosine (5hmC) has triggered an avalanche of studies aiming to resolve the role of 5hmC in gene regulation if any. Hitherto, TET1 is reported to bind to CpG‐island (CGI) and bivalent promoters in mouse embryonic stem cells, whereas binding at DNAseI hypersensitive sites (HS) had escaped previous analysis. Significant enrichment/accumulation of 5hmC but not 5mC can indeed be detected at bivalent promoters and at DNaseI‐HS. Surprisingly, however, 5hmC is not detected or present at very low levels at CGI promoters notwithstanding the presence of TET1. Our meta‐analysis of DNA methylation profiling points to potential issues with regard to the various methodologies that are part of the toolbox used to detect 5mC and 5hmC. Discrepancies between published studies and technical limitations prevent an unambiguous assignment of 5hmC as a ‘true’ epigenetic mark, that is, read and interpreted by other factors and/or as a transiently accumulating intermediary product of the conversion of 5mC to unmodified cytosines.     

Epigenetic silenced miR‐125a‐5p could be self‐activated through targeting Suv39H1 in gastric cancer

Emerging evidence suggests that microRNAs (miRNAs) serve an important role in tumorigenesis and development. Although the low expression of miR‐125a‐5p in gastric cancer has been reported, the underlying mechanism remains unknown. In the current study, the low expression of miR‐125a‐5p in gastric cancer was verified in paired cancer tissues and adjacent non‐tumour tissues. Furthermore, the GC islands in the miR‐125a‐5p region were hypermethylated in the tumour tissues. And the hypermethylation was negatively correlated with the miR‐125a‐5p expression. Target gene screening showed that the histone methyltransferase Suv39H1 was one of the potential target genes. In vitro studies showed that miR‐125a‐5p could directly suppress the Suv39H1 expression and decrease the H3K9me3 levels. On the other hand, the Suv39H1 could induce demethylation of miR‐125a‐5p, resulting in re‐activation of miR‐125a‐5p. What is more, overexpessing miR‐125a‐5p could also self‐activate the silenced miR‐125a‐5p in gastric cancer cells, which suppressed cell migration, invasion and proliferation in vitro and inhibited cancer progression in vivo. Thus, we uncovered here that the epigenetic silenced miR‐125a‐5p could be self‐activated through targeting Suv39H1 in gastric cancer, suggesting that miR‐125a‐5p might be not only the potential prognostic value as a tumour biomarker but also potential therapeutic targets in gastric cancer.     

Associations of genome-wide cell-free DNA fragmentation profiles with blood biochemical and hematological parameters in healthy individuals

Cell-free DNA (cfDNA), as a non-invasive approach, has been introduced in a wide range of applications, including cancer diagnosis/ monitoring, prenatal testing, and transplantation monitoring. Yet, studies of cfDNA fragmentomics in physiological conditions are lacking. In this study, we aim to explore the correlation of fragmentation patterns of cfDNA with blood biochemical and hematological parameters in healthy individuals. We addressed the impact of physiological variables and abnormal blood biochemical and hematological parameters on cfDNA fragment size distribution. We also figured and validated that hematological inflammation markers, including leukocyte, lymphocyte, neutrophil, and platelet distribution width as well as aspartate transaminase levels were significantly correlated with the genome-wide cfDNA fragmentation pattern. Our findings suggest that cfDNA fragmentation profiles were associated with physiological parameters related to cardiovascular risk factors, inflammatory response and hepatocyte injury, which may provide insights for further research on the potential role of cfDNA fragmentation in diagnosis and monitor of several disease.     

Caveolin‐1 as a promoter of tumour spreading: when,how, where and why

Caveolae are non‐clathrin invaginations of the plasma membrane in most cell types; they are involved in signalling functions and molecule trafficking, thus modulating several biological functions, including cell growth, apoptosis and angiogenesis. The major structural protein in caveolae is caveolin‐1, which is known to act as a key regulator in cancer onset and progression through its role as a tumour suppressor. Caveolin‐1 can also promote cell proliferation, survival and metastasis as well as chemo‐ and radioresistance. Here, we discuss recent findings and novel concepts that support a role for caveolin‐1 in cancer development and its distant spreading. We also address the potential application of caveolin‐1 in tumour therapy and diagnosis.     

Phenethyl isothiocyanate potentiates anti‐tumour effect of doxorubicin through Akt‐dependent pathway

The present study aims to investigate the in vivo and in vitro anti‐tumour properties of phenethyl isothiocyanate (PEITC) alone and in combination with doxorubicin (Dox). The anti‐tumour activity was evaluated in vitro by MTT assay using cultured human breast cancer cell line (MCF‐7) and human hepatoma cell line (HepG‐2) cell lines. In vivo, Ehrlich solid tumour model was used. Tumour volume, weight and antioxidant parameters were determined. Immunohistochemistry analysis for active (cleaved) caspase‐3 was also performed. We tested the effect of PEITC treatment on pAkt/Akt ratio, NF‐κB p65 DNA binding activity and caspase‐9 enzyme activity in both MCF‐7 and HepG‐2 cell lines. Effect of PEITC treatment on cell migration was assessed by wound healing assay. PEITC and/or Dox treatment significantly inhibited solid tumour volume and tumour weight when compared with control mice. PEITC treatment significantly reduced oxidative stress caused by Dox treatment as indicated by significant increase in total antioxidant capacity and decrease in malondialdehyde level. Microscopic examination of tumour tissues showed a significant increase in active (cleaved) caspase‐3 expression in PEITC and/or Dox treated groups. PEITC showed a dose‐dependent inhibition of MCF‐7 and HepG‐2 cellular viability. PEITC inhibited Akt and NF‐κB activation and increased caspase‐9 activity in a dose‐dependent manner. PEITC treatment effectively inhibited both MCF‐7 and HepG‐2 cell migration. We can conclude that PEITC acts via multiple molecular targets to elicit anti‐carcinogenic activity. PEITC/Dox combination therapy might be a potential novel strategy, which may benefit patients with breast and liver cancers. Copyright © 2015 John Wiley & Sons, Ltd.