No abstract

BackgroundCirculating cell-free DNA (cfDNA) has recently been recognized as a resource for biomarkers of cancer progression, treatment response, and drug resistance. However, few have demonstrated the usefulness of cfDNA for early detection of cancer. Although aberrant DNA methylation in cfDNA has been reported for more than a decade, its diagnostic accuracy remains unsatisfactory for cancer screening. Thus, the aim of the present study was to develop a highly sensitive cfDNA-based system for detection of primary breast cancer (BC) using epigenetic biomarkers and digital PCR technology.MethodsArray-based genome-wide DNA methylation analysis was performed using 56 microdissected breast tissue specimens, 34 cell lines, and 29 blood samples from healthy volunteers (HVs). Epigenetic markers for BC detection were selected, and a droplet digital methylation-specific PCR (ddMSP) panel with the selected markers was established. The detection model was constructed by support vector machine and evaluated using cfDNA samples.ResultsThe methylation array analysis identified 12 novel epigenetic markers (JAK3, RASGRF1, CPXM1, SHF, DNM3, CAV2, HOXA10, B3GNT5, ST3GAL6, DACH1, P2RX3, and chr8:23572595) for detecting BC. We also selected four internal control markers (CREM, GLYATL3, ELMOD3, and KLF9) that were identified as infrequently altered genes using a public database. A ddMSP panel using these 16 markers was developed and detection models were constructed with a training dataset containing cfDNA samples from 80 HVs and 87 cancer patients. The best detection model adopted four methylation markers (RASGRF1, CPXM1, HOXA10, and DACH1) and two parameters (cfDNA concentration and the mean of 12 methylation markers), and, and was validated in an independent dataset of 53 HVs and 58 BC patients. The area under the receiver operating characteristic curve for cancer-normal discrimination was 0.916 and 0.876 in the training and validation dataset, respectively. The sensitivity and the specificity of the model was 0.862 (stages 0-I 0.846, IIA 0.862, IIB-III 0.818, metastatic BC 0.935) and 0.827, respectively.ConclusionOur epigenetic-marker-based system distinguished BC patients from HVs with high accuracy. As detection of early BC using this system was comparable with that of mammography screening, this system would be beneficial as an optional method of screening for BC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0788-z) contains supplementary material, which is available to authorized users.

Baseline CEC, in particular CD34(+)CEC, counts and the CD34 positive rate might be useful for the prediction of treatment response of preoperative chemotherapy in patients with operable breast cancer.

17During metastasis, cancer cell migration is enhanced. However, the mechanisms 18 underlying this process remain elusive. Here, we addressed this issue by functionally 19 analyzing the transcription factor Sal-like 4 (SALL4) in basal-like breast cancer cells. 20Loss-of-function studies of SALL4 showed that this transcription factor is required for 21 the spindle-shaped morphology and the enhanced migration of cancer cells. SALL4 also 22 up-regulated integrin gene expression. The impaired cell migration observed in SALL4 23 knockdown cells was restored by overexpression of integrin α6 and β1. In addition, we 24 clarified that integrin α6 and β1 formed a heterodimer. At the molecular level, loss of 25 the SALL4 -integrin α6β1 network lost focal adhesion dynamics, which impairs cell 26 migration. Over-activation of Rho is known to inhibit focal adhesion dynamics. We 27 observed that SALL4 knockdown cells exhibited over-activation of Rho. Aberrant Rho The SALL4 -integrin α6β1 network is required for a spindle-shaped morphology. 39The SALL4 -integrin α6β1 network activates focal adhesion dynamics. 40The SALL4 -integrin α6β1 network modulates Rho activation for cell migration. 41 42

Summary Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) has been demonstrated to result in various stress-related diseases, including diabetes mellitus. Deficiency of cellular nicotinamide adenine dinucleotide (NAD ϩ ) content, consumed by PARP-1 to add ADP-ribose moieties onto target proteins, contributes to pathophysiological conditions. Adenosine thiamine triphosphate (AThTP) exists in small amounts in mammals; however, the function(s) of this metabolite remains unresolved. The structure of AThTP resembles NAD Substantial recent experimental studies demonstrate beneficial impacts of high-dose thiamine on diabetic complications, such as diabetic retinopathy, diabetic nephropathy, diabetic neuropathy and diabetic cardiomyopathy ( 1-4 ). However, the pharmacological relevance of high-dose thiamine treatments remains unknown.Chronic hyperglycemia results in diabetic complications in target organs. The pathogenic effect of high glucose is, at least partially, mediated to a significant extent through increased production of reactive oxygen species and reactive nitrogen species and subsequent oxidative stress (reviewed in Evans et al. ( 5 )). Increased oxidative stress activates the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 activation depletes its substrate, nicotinamide adenine dinucleotide (NAD ϩ ), and also covalently attaches branched nucleic acid-like polymers of poly(ADP-ribose) to various acceptor proteins (reviewed in Kiss and Szabó ( 6 )). A covalently attached ADP-ribose polymer, poly(ADP-ribosylation, affects the function of target proteins. The involvement of PARP-1 activation in the pathogenesis of diabetes and its complications has recently been emphasized by both in vivo and in vitro studies (reviewed in Pacher and Szabó ( 7 ), and Szabó ( 8 )). NAD ϩ , used as substrate for PARP-1, consists of two nucleotides joined through their phosphate groups, with one nucleotide containing an adenine base and the other containing nicotinamide.Adenosine thiamine triphosphate (AThTP), a new thiamine derivative, was recently identified in Escherichia coli ( 9 ), followed by the identification in small amounts in mouse brain, heart, skeletal muscle, liver and kidneys ( 10 ). AThTP is composed of two molecules, an adenine base and thiamine, which are joined through phosphate groups. The structure of AThTP appears to closely resemble NAD ϩ . Although the biological role of AThTP is unknown, the existence of noncoenzyme functions of thiamine derivatives has been speculated (11)(12)(13)(14).In the context of 1) structural resemblance of AThTP to NAD ϩ , 2) the experimental evidence implicating PARP-1 as a causative factor in the pathogenesis of diabetes and diabetic complications in vitro and in vivo (reviewed in Szabó ( 8 )), and 3) beneficial effects of highdose thiamine on diabetic complications, we hypothesized that AThTP could interact with PARP-1 and mod-E-mail: t.tanaka.md@gmail.com Abbreviations: AThTP, adenosine thiamine triphosphate; NAD ϩ , nicotinamide adenine dinucleotide; PARP...