Exosome-Mediated Paracrine Signaling Unveils miR-1246 as a Driver of Aggressiveness in Fusion-Negative Rhabdomyosarcoma.

Rhabdomyosarcoma is a pediatric cancer associated with aggressiveness and a tendency to develop metastases. Fusion-negative rhabdomyosarcoma (FN-RMS) is the most commonly occurring subtype of RMS, where metastatic disease can hinder treatment success and decrease survival rates. RMS-derived exosomes were previously demonstrated to be enriched with miRNAs, including miR-1246, possibly contributing to disease aggressiveness. We aimed to decipher the functional impact of exosomal miR-1246 on recipient cells and its role in promoting aggressiveness. Treatment of normal fibroblasts with FN-RMS-derived exosomes resulted in a significant uptake of miR-1246 paired with an increase in cell proliferation, migration, and invasion. In turn, delivery of miR-1246-mimic lipoplexes promoted fibroblast proliferation, migration, and invasion in a similar manner. Conversely, when silencing miR-1246 in FN-RMS cells, the resulting derived exosomes demonstrated reversed effects on recipient cells' phenotype. Delivery of exosomal miR-1246 targets GSK3ß and promotes ß-catenin nuclear accumulation, suggesting a deregulation of the Wnt pathway, known to be important in tumor progression. Finally, a pilot clinical study highlighted, for the first time, the presence of high exosomal miR-1246 levels in RMS patients' sera. Altogether, our results demonstrate that exosomal miR-1246 has the potential to alter the tumor microenvironment of FN-RMS cells, suggesting its potential role in promoting oncogenesis.

Aflatoxin B1 and Epstein-Barr virus-induced CCL22 expression stimulates B cell infection.

Epstein-Barr Virus (EBV) infects more than 90% of the adult population worldwide. EBV infection is associated with Burkitt lymphoma (BL) though alone is not sufficient to induce carcinogenesis implying the involvement of co-factors. BL is endemic in African regions faced with mycotoxins exposure. Exposure to mycotoxins and oncogenic viruses has been shown to increase cancer risks partly through the deregulation of the immune response. A recent transcriptome profiling of B cells exposed to aflatoxin B1 (AFB1) revealed an upregulation of the Chemokine ligand 22 (CCL22) expression although the underlying mechanisms were not investigated. Here, we tested whether mycotoxins and EBV exposure may together contribute to endemic BL (eBL) carcinogenesis via immunomodulatory mechanisms involving CCL22. Our results revealed that B cells exposure to AFB1 and EBV synergistically stimulated CCL22 secretion via the activation of Nuclear Factor-kappa B pathway. By expressing EBV latent genes in B cells, we revealed that elevated levels of CCL22 result not only from the expression of the latent membrane protein LMP1 as previously reported but also from the expression of other viral latent genes. Importantly, CCL22 overexpression resulting from AFB1-exposure in vitro increased EBV infection through the activation of phosphoinositide-3-kinase pathway. Moreover, inhibiting CCL22 in vitro and in humanized mice in vivo limited EBV infection and decreased viral genes expression, supporting the notion that CCL22 overexpression plays an important role in B cell infection. These findings unravel new mechanisms that may underpin eBL development and identify novel pathways that can be targeted in drug development.

At the crossroads of fertility and metabolism: the importance of AMPK-dependent signaling in female infertility associated with hyperandrogenism.

STUDY QUESTION: What biological processes are linked to the signaling of the energy sensor 5'-AMP-activated protein kinase (AMPK) in mouse and human granulosa cells (GCs)? SUMMARY ANSWER: The lack of α1AMPK in GCs impacted cell cycle, adhesion, lipid metabolism and induced a hyperandrogenic response. WHAT IS KNOWN ALREADY: AMPK is expressed in the ovarian follicle, and its activation by pharmacological medications, such as metformin, inhibits the production of steroids. Polycystic ovary syndrome (PCOS) is responsible for infertility in approximately 5-20% of women of childbearing age and possible treatments include reducing body weight, improving lifestyle and the administration of a combination of drugs to improve insulin resistance, such as metformin. STUDY DESIGN, SIZE, DURATION: AMPK signaling was evaluated by analyzing differential gene expression in immortalized human granulosa cells (KGNs) with and without silencing α1AMPK using CRISPR/Cas9. In vivo studies included the use of a α1AMPK knock-out mouse model to evaluate the role of α1AMPK in folliculogenesis and fertility. Expression of α1AMPK was evaluated in primary human granulosa-luteal cells retrieved from women undergoing IVF with and without a lean PCOS phenotype (i.e. BMI: 18-25 kg/m2). PARTICIPANTS/MATERIALS, SETTING, METHODS: α1AMPK was disrupted in KGN cells and a transgenic mouse model. Cell viability, proliferation and metabolism were evaluated. Androgen production was evaluated by analyzing protein levels of relevant enzymes in the steroid pathway by western blots, and steroid levels obtained from in vitro and in vivo models by mass spectrometry. Differential gene expression in human GC was obtained by RNA sequencing. Analysis of in vivo murine folliculogenesis was performed by histology and immunochemistry, including evaluation of the anti-Müllerian hormone (AMH) marker. The α1AMPK gene expression was evaluated by quantitative RT-PCR in primary GCs obtained from women with the lean PCOS phenotype (n = 8) and without PCOS (n = 9). MAIN RESULTS AND THE ROLE OF CHANCE: Silencing of α1AMPK in KGN increased cell proliferation (P < 0.05 versus control, n = 4), promoted the use of fatty acids over glucose, and induced a hyperandrogenic response resulting from upregulation of two of the enzymes involved in steroid production, namely 3ß-hydroxysteroid dehydrogenase (3ßHSD) and P450 side-chain cleavage enzyme (P450scc) (P < 0.05, n = 3). Female mice deficient in α1AMPK had a 30% decrease in their ovulation rate (P < 0.05, n = 7) and litter size, a hyperandrogenic response (P < 0.05, n = 7) with higher levels of 3ßHSD and p450scc levels in the ovaries, and an increase in the population of antral follicles (P < 0.01, n = 10) compared to controls. Primary GCs from lean women with PCOS had lower α1AMPK mRNA expression levels than the control group (P < 0.05, n = 8-9). LARGE SCALE DATA: The FastQ files and metadata were submitted to the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB46048. LIMITATIONS, REASONS FOR CAUTION: The human KGN is a not fully differentiated, transformed cell line. As such, to confirm the role of AMPK in GC and the PCOS phenotype, this model was compared to two others: an α1AMPK transgenic mouse model and primary differentiated granulosa-lutein cells from non-obese women undergoing IVF (with and without PCOS). A clear limitation is the small number of patients with PCOS utilized in this study and that the collection of human GCs was performed after hormonal stimulation. WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal that AMPK is directly involved in steroid production in human GCs. In addition, AMPK signaling was associated with other processes frequently reported as dysfunctional in PCOS models, such as cell adhesion, lipid metabolism and inflammation. Silencing of α1AMPK in KGN promoted folliculogenesis, with increases in AMH. Evaluating the expression of the α1AMPK subunit could be considered as a marker of interest in infertility cases related to hormonal imbalances and metabolic disorders, including PCOS. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by the Institut National de la Recherche Agronomique (INRA) and the national programme « FERTiNERGY ¼ funded by the French National Research Agency (ANR). The authors report no intellectual or financial conflicts of interest related to this work. R.K. is identified as personnel of the International Agency for Research on Cancer/World Health Organization. R.K. alone is responsible for the views expressed in this article and she does not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization. TRIAL REGISTRATION NUMBER: N/A.

Epigenetic Alteration of the Cancer-Related Gene TGFBI in B Cells Infected with Epstein-Barr Virus and Exposed to Aflatoxin B1: Potential Role in Burkitt Lymphoma Development.

Burkitt lymphoma (BL) is a malignant B cell neoplasm that accounts for almost half of pediatric cancers in sub-Saharan African countries. Although the BL endemic prevalence is attributable to the combination of Epstein-Barr virus (EBV) infection with malaria and environmental carcinogens exposure, such as the food contaminant aflatoxin B1 (AFB1), the molecular determinants underlying the pathogenesis are not fully understood. Consistent with the role of epigenetic mechanisms at the interface between the genome and environment, AFB1 and EBV impact the methylome of respectively leukocytes and B cells specifically. Here, we conducted a thorough investigation of common epigenomic changes following EBV or AFB1 exposure in B cells. Genome-wide DNA methylation profiling identified an EBV-AFB1 common signature within the TGFBI locus, which encodes for a putative tumor suppressor often altered in cancer. Subsequent mechanistic analyses confirmed a DNA-methylation-dependent transcriptional silencing of TGFBI involving the recruitment of DNMT1 methyltransferase that is associated with an activation of the NF-κB pathway. Our results reveal a potential common mechanism of B cell transformation shared by the main risk factors of endemic BL (EBV and AFB1), suggesting a key determinant of disease that could allow the development of more efficient targeted therapeutic strategies.

DNA Methylome Changes of Muscle- and Neuronal-Related Processes Precede Bladder Cancer Invasiveness.

Bladder cancer (BC) is the ninth leading cause of cancer death with one of the highest recurrence rates among all cancers. One of the main risks for BC development is exposure to nitrosamines present in tobacco smoke or in other products. Aberrant epigenetic (DNA methylation) changes accompanied by deregulated gene expression are an important element of cancer pathogenesis. Therefore, we aimed to determine DNA methylation signatures and their impacts on gene expression in mice treated with N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), a carcinogen similar to compounds found in tobacco smoke. Following BBN administration mice developed non-invasive or invasive bladder cancers. Surprisingly, muscle- and neuronal-related pathways emerged as the most affected in those tumors. Hypo- and hypermethylation changes were present within non-invasive BC, across CpGs mapping to the genes involved in muscle- and neuronal-related pathways, however, methylation differences were not sufficient to affect the expression of the majority of associated genes. Conversely, invasive tumors displayed hypermethylation changes that were linked with alterations in gene expression profiles. Together, these findings indicate that bladder cancer progression could be revealed through methylation profiling at the pre-invasive cancer stage that could assist monitoring of cancer patients and guide novel therapeutic approaches.

In Utero Exposure to Metformin Reduces the Fertility of Male Offspring in Adulthood.

Metformin is a drug used for the treatment of type 2 diabetes and disorders associated with insulin resistance. Metformin is also used in the treatment of pregnancy disorders such as gestational diabetes. However, the consequences of foetal exposure to metformin on the fertility of exposed offspring remain poorly documented. In this study, we investigated the effect of in utero metformin exposure on the fertility of female and male offspring. We observed that metformin is detectable in the blood of the mother and in amniotic fluid and blood of the umbilical cord. Metformin was not measurable in any tissues of the embryo, including the gonads. The effect of metformin exposure on offspring was sex specific. The adult females that had been exposed to metformin in utero presented no clear reduction in fertility. However, the adult males that had been exposed to metformin during foetal life exhibited a 30% reduction in litter size compared with controls. The lower fertility was not due to a change in sperm production or the motility of sperm. Rather, the phenotype was due to lower sperm head quality - significantly increased spermatozoa head abnormality with greater DNA damage - and hypermethylation of the genomic DNA in the spermatozoa associated with lower expression of the ten-eleven translocation methylcytosine dioxygenase 1 (TET1) protein. In conclusion, while foetal metformin exposure did not dramatically alter gonad development, these results suggest that metabolic modification by metformin during the foetal period could change the expression of epigenetic regulators such as Tet1 and perturb the genomic DNA in germ cells, changes that might contribute to a reduced fertility.

Epigenetic remodelling of enhancers in response to estrogen deprivation and re-stimulation.

Estrogen hormones are implicated in a majority of breast cancers and estrogen receptor alpha (ER), the main nuclear factor mediating estrogen signaling, orchestrates a complex molecular circuitry that is not yet fully elucidated. Here, we investigated genome-wide DNA methylation, histone acetylation and transcription after estradiol (E2) deprivation and re-stimulation to better characterize the ability of ER to coordinate gene regulation. We found that E2 deprivation mostly resulted in DNA hypermethylation and histone deacetylation in enhancers. Transcriptome analysis revealed that E2 deprivation leads to a global down-regulation in gene expression, and more specifically of TET2 demethylase that may be involved in the DNA hypermethylation following short-term E2 deprivation. Further enrichment analysis of transcription factor (TF) binding and motif occurrence highlights the importance of ER connection mainly with two partner TF families, AP-1 and FOX. These interactions take place in the proximity of E2 deprivation-mediated differentially methylated and histone acetylated enhancers. Finally, while most deprivation-dependent epigenetic changes were reversed following E2 re-stimulation, DNA hypermethylation and H3K27 deacetylation at certain enhancers were partially retained. Overall, these results show that inactivation of ER mediates rapid and mostly reversible epigenetic changes at enhancers, and bring new insight into early events, which may ultimately lead to endocrine resistance.

Genome-Wide DNA Methylation Profiling of Esophageal Squamous Cell Carcinoma from Global High-Incidence Regions Identifies Crucial Genes and Potential Cancer Markers.

Epigenetic mechanisms such as aberrant DNA methylation (DNAme) are known to drive esophageal squamous cell carcinoma (ESCC), yet they remain poorly understood. Here, we studied tumor-specific DNAme in ESCC cases from nine high-incidence countries of Africa, Asia, and South America. Infinium MethylationEPIC array was performed on 108 tumors and 51 normal tissues adjacent to the tumors (NAT) in the discovery phase, and targeted pyrosequencing was performed on 132 tumors and 36 NAT in the replication phase. Top genes for replication were prioritized by weighting methylation results using RNA-sequencing data from The Cancer Genome Atlas and GTEx and validated by qPCR. Methylome analysis comparing tumor and NAT identified 6,796 differentially methylated positions (DMP) and 866 differential methylated regions (DMR), with a 30% methylation (Δß) difference. The majority of identified DMPs and DMRs were hypermethylated in tumors, particularly in promoters and gene-body regions of genes involved in transcription activation. The top three prioritized genes for replication, PAX9, SIM2, and THSD4, had similar methylation differences in the discovery and replication sets. These genes were exclusively expressed in normal esophageal tissues in GTEx and downregulated in tumors. The specificity and sensitivity of these DNAme events in discriminating tumors from NAT were assessed. Our study identified novel, robust, and crucial tumor-specific DNAme events in ESCC tumors across several high-incidence populations of the world. Methylome changes identified in this study may serve as potential targets for biomarker discovery and warrant further functional characterization. SIGNIFICANCE: This largest genome-wide DNA methylation study on ESCC from high-incidence populations of the world identifies functionally relevant and robust DNAme events that could serve as potential tumor-specific markers. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/10/2612/F1.large.jpg.

Pan-cancer multi-omics analysis and orthogonal experimental assessment of epigenetic driver genes.

The recent identification of recurrently mutated epigenetic regulator genes (ERGs) supports their critical role in tumorigenesis. We conducted a pan-cancer analysis integrating (epi)genome, transcriptome, and DNA methylome alterations in a curated list of 426 ERGs across 33 cancer types, comprising 10,845 tumor and 730 normal tissues. We found that, in addition to mutations, copy number alterations in ERGs were more frequent than previously anticipated and tightly linked to expression aberrations. Novel bioinformatics approaches, integrating the strengths of various driver prediction and multi-omics algorithms, and an orthogonal in vitro screen (CRISPR-Cas9) targeting all ERGs revealed genes with driver roles within and across malignancies and shared driver mechanisms operating across multiple cancer types and hallmarks. This is the largest and most comprehensive analysis thus far; it is also the first experimental effort to specifically identify ERG drivers (epidrivers) and characterize their deregulation and functional impact in oncogenic processes.

Coordination of germ layer lineage choice by TET1 during primed pluripotency.

Gastrulation in the early postimplantation stage mammalian embryo begins when epiblast cells ingress to form the primitive streak or develop as the embryonic ectoderm. The DNA dioxygenase Tet1 is highly expressed in the epiblast and yet continues to regulate lineage specification during gastrulation when its expression is diminished. Here, we show how Tet1 plays a pivotal role upstream of germ layer lineage bifurcation. During the transition from naive pluripotency to lineage priming, a global reconfiguration redistributes Tet1 from Oct4-cobound promoters to distal regulatory elements at lineage differentiation genes, which are distinct from high-affinity sites engaged by Oct4. An altered chromatin landscape in Tet1-deficient primed epiblast-like cells is associated with enhanced Oct4 expression and binding to Nodal and Wnt target genes, resulting in collaborative signals that enhance mesendodermal and inhibit neuroectodermal gene expression during lineage segregation. A permissive role for Tet1 in neural fate induction involves Zic2-dependent engagement at neural target genes at lineage priming, is dependent on the signaling environment during gastrulation, and impacts neural tube closure after gastrulation. Our findings provide mechanistic information for epigenetic integration of pluripotency and signal-induced differentiation cues.

Regulatory Dynamics of Tet1 and Oct4 Resolve Stages of Global DNA Demethylation and Transcriptomic Changes in Reprogramming.

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) involves the reactivation of endogenous pluripotency genes and global DNA demethylation, but temporal resolution of these events using existing markers is limited. Here, we generate murine transgenic lines harboring reporters for the 5-methylcytosine dioxygenase Tet1 and for Oct4. By monitoring dual reporter fluorescence during pluripotency entry, we identify a sequential order of Tet1 and Oct4 activation by proximal and distal regulatory elements. Full Tet1 activation marks an intermediate stage that accompanies predominantly repression of somatic genes, preceding full Oct4 activation, and distinguishes two waves of global DNA demethylation that target distinct genomic features but are uncoupled from transcriptional changes. Tet1 knockout shows that TET1 contributes to both waves of demethylation and activates germline regulatory genes in reprogramming intermediates but is dispensable for Oct4 reactivation. Our dual reporter system for time-resolving pluripotency entry thus refines the molecular roadmap of iPSC maturation.

Metformin in Reproductive Biology.

Initially produced in Europe in 1958, metformin is still one of the most widely prescribed drugs to treat type II diabetes and other comorbidities associated with insulin resistance. Metformin has been shown to improve fertility outcomes in females with insulin resistance associated with polycystic ovary syndrome (PCOS) and in obese males with reduced fertility. Metformin treatment reinstates menstrual cyclicity, decreases the incidence of cesareans, and limits the number of premature births. Notably, metformin reduces steroid levels in conditions associated with hyperandrogenism (e.g., PCOS and precocious puberty) in females and improves fertility of adult men with metabolic syndrome through increased testosterone production. While the therapeutical use of metformin is considered to be safe, in the last 10 years some epidemiological studies have described phenotypic differences after prenatal exposure to metformin. The goals of this review are to briefly summarize the current knowledge on metformin focusing on its effects on the female and male reproductive organs, safety concerns, including the potential for modulating fetal imprinting via epigenetics.

Lineage-specific functions of TET1 in the postimplantation mouse embryo.

The mammalian TET enzymes catalyze DNA demethylation. While they have been intensely studied as major epigenetic regulators, little is known about their physiological roles and the extent of functional redundancy following embryo implantation. Here we define non-redundant roles for TET1 at an early postimplantation stage of the mouse embryo, when its paralogs Tet2 and Tet3 are not detectably expressed. TET1 regulates numerous genes defining differentiation programs in the epiblast and extraembryonic ectoderm. In epiblast cells, TET1 demethylates gene promoters via hydroxymethylation and maintains telomere stability. Surprisingly, TET1 represses a majority of epiblast target genes independently of methylation changes, in part through regulation of the gene encoding the transcriptional repressor JMJD8. Dysregulated gene expression in the absence of TET1 causes embryonic defects, which are partially penetrant in an inbred strain but fully lethal in non-inbred mice. Collectively, our study highlights an interplay between the catalytic and non-catalytic activities of TET1 that is essential for normal development.

PDGFRα+ Cells in Embryonic Stem Cell Cultures Represent the In Vitro Equivalent of the Pre-implantation Primitive Endoderm Precursors.

In early mouse pre-implantation development, primitive endoderm (PrE) precursors are platelet-derived growth factor receptor alpha (PDGFRα) positive. Here, we demonstrated that cultured mouse embryonic stem cells (mESCs) express PDGFRα heterogeneously, fluctuating between a PDGFRα+ (PrE-primed) and a platelet endothelial cell adhesion molecule 1 (PECAM1)-positive state (epiblast-primed). The two surface markers can be co-detected on a third subpopulation, expressing epiblast and PrE determinants (double-positive). In vitro, these subpopulations differ in their self-renewal and differentiation capability, transcriptional and epigenetic states. In vivo, double-positive cells contributed to epiblast and PrE, while PrE-primed cells exclusively contributed to PrE derivatives. The transcriptome of PDGFRα+ subpopulations differs from previously described subpopulations and shows similarities with early/mid blastocyst cells. The heterogeneity did not depend on PDGFRα but on leukemia inhibitory factor and fibroblast growth factor signaling and DNA methylation. Thus, PDGFRα+ cells represent the in vitro counterpart of in vivo PrE precursors, and their selection from cultured mESCs yields pure PrE precursors.

Cytokeratin-20 and Survivin-Expressing Circulating Tumor Cells Predict Survival in Metastatic Colorectal Cancer Patients by a Combined Immunomagnetic qRT-PCR Approach.

Circulating tumor cells (CTC) express epithelial and stem cell-like genes, though current approved detection methods mainly use epithelial markers. We optimized a CTC isolation method that could capture their molecular heterogeneity and predict overall survival (OS) in metastatic colorectal cancer (mCRC) patients receiving various chemotherapy regimens. We combined immunomagnetic enrichment of CD45-negative, EpCAM-positive circulating cancer cells with qRT-PCR amplification of CK20 and survivin expression in 88 mCRC patients and 20 healthy controls. We then evaluated the prognostic value of baseline CTC CK20 and survivin expression in mCRC patients. The presence of elevated CTC CK20 or survivin expression distinguished mCRC patients from controls with sufficient sensitivity (79.6%) and specificity (85%). In univariate analysis, patients with high CTC-CK20 expression (9 vs. 33.2+ months, log-rank P < 0.001) or high CTC-survivin expression (10 vs. 33.2+ months, log-rank P = 0.032) had a significantly worse median OS than those with low expression of either marker. In multivariable analysis, the high CTC-CK20 group had significantly shortened OS (HR, 3.11; adjusted P = 0.01), and there was a trend toward inferior OS in the high CTC-survivin group (HR, 1.76; adjusted P = 0.099). Patients with either high CTC CK20 or survivin expression had inferior OS compared with those with low expression of both markers (HR, 4.39; 95% confidence interval, 1.56-12.35; adjusted P = 0.005). Colorectal cancer CTCs can be reliably isolated using epithelial and stem cell markers. CTC CK20 and survivin expression may effectively predict OS in mCRC patients receiving chemotherapy.

Genetic variations in angiopoietin and pericyte pathways and clinical outcome in patients with resected colorectal liver metastases.

BACKGROUND: Genes involved in the angiopoietin and pericyte pathways may become escape mechanisms under antivascular endothelial growth factor (anti-VEGF) therapy. The authors investigated whether variations within genes in these pathways are associated with clinical outcome in patients with colorectal liver metastases who undergo liver resection and receive perioperative, bevacizumab-based chemotherapy. METHODS: Single nucleotide polymorphisms (SNPs) in 9 genes (angiopoietin-1 [ANGPT1]; ANGPT2; TEK tyrosine kinase, endothelial [TEK]; platelet-derived growth factor ß [PDGFB]; ß-type platelet-derived growth factor receptor [PDGFRB]; insulin-like growth factor 1 [IGF1]; transforming growth factor ß1 [TGFB1]; RalA binding protein 1 [RALBP1]; and regulator of G-protein signaling 5 [RGS5]) were analyzed in samples of genomic DNA from 149 patients and were evaluated for associations with clinical outcome. RESULTS: RALBP1 reference SNP 329007 (rs329007) A>G resulted in a significant difference in recurrence-free survival (A/A genotype, 14.0 months; A/G or G/G genotype, 9.2 months; hazard ratio [HR], 1.60; P = .024). PDGFB rs1800818 A>G was associated with 3-year overall survival rates (A/A genotype, 78%; A/G genotype, 69%; [HR 1.37]; G/G genotype, 53%; [HR 2.12]; P = .048). In multivariate analysis, RALBP1 rs329007 A>G remained significant (HR, 1.99; P = .002). PDGFB rs1800818 A>G and RALBP1 rs329007 A>G were correlated with radiologic response (A/A or A/G genotype, 86%; G/G genotype, 71% [P = .042]; A/A genotype, 78%; A/G or G/G genotype, 94% [P = .018], respectively). RALBP1 rs329007 A>G demonstrated significantly different rates of histologic response (A/A genotype: major histologic response, 35%; partial histologic response, 34%; no histologic response, 30%; A/G or G/G genotype: 46%, 13%, and 41%, respectively; P = .029). Recursive partitioning analysis revealed that ANGPT2 rs2442599 T>C and RALBP1 rs329007 A>G were the main SNPs that predicted histologic response and recurrence-free survival, whereas PDGFB rs1800818 A>G was the leading SNP that predicted overall survival. ANGPT2 rs2916702 C>T and rs2442631 G>A were significantly associated with the probability of achieving a cure. CONCLUSIONS: The current data suggest that variations in genes involved in the angiopoietin and pericyte pathways may be predictive and/or prognostic biomarkers in patients with resected colorectal liver metastases who receive bevacizumab-based chemotherapy.

Dynamic switching of active promoter and enhancer domains regulates Tet1 and Tet2 expression during cell state transitions between pluripotency and differentiation.

The Tet 5-methylcytosine dioxygenases catalyze DNA demethylation by producing 5-hydroxymethylcytosine and further oxidized products. Tet1 and Tet2 are highly expressed in mouse pluripotent cells and downregulated to different extents in somatic cells, but the transcriptional mechanisms are unclear. Here we defined the promoter and enhancer domains in Tet1 and Tet2. Within a 15-kb "superenhancer" of Tet1, there are two transcription start sites (TSSs) with different activation patterns during development. A 6-kb promoter region upstream of the distal TSS is highly active in naive pluripotent cells, autonomously reports Tet1 expression in a transgenic system, and rapidly undergoes DNA methylation and silencing upon differentiation in cultured cells and native epiblast. A second TSS downstream, associated with a constitutively weak CpG-rich promoter, is activated by a neighboring enhancer in naive embryonic stem cells (ESCs) and primed epiblast-like cells (EpiLCs). Tet2 has a CpG island promoter with pluripotency-independent activity and an ESC-specific distal intragenic enhancer; the latter is rapidly downregulated in EpiLCs. Our study reveals distinct modes of transcriptional regulation at Tet1 and Tet2 during cell state transitions of early development. New transgenic reporters using Tet1 and Tet2 cis-regulatory domains may serve to distinguish nuanced changes in pluripotent states and the underlying epigenetic variations.

Prognostic impact of the c-MET polymorphism on the clinical outcome in locoregional gastric cancer patients.

OBJECTIVE: Dysregulation of the c-MET signaling pathway results from various molecular mechanisms including mutation, amplification, and overexpression. Overexpression and amplification of c-MET have been correlated with poor clinical outcome in gastric cancer, whereas the associations between c-MET polymorphisms and prognosis have not been well defined. We examined the prognostic impact of functional polymorphisms of the MET gene on clinical outcome in gastric cancer. METHODS: Candidate polymorphisms of the MET gene were analyzed by PCR-based direct sequencing for the associations with clinical outcome across three independent cohorts, including 161 Japanese, 101 US, and 63 Austrian patients, with locoregional gastric cancer, treated with surgery. RESULTS: The univariable analysis showed that patients with any G (A/G or G/G genotype) allele of MET rs40239 had significantly longer disease-free survival and overall survival compared with those with the AA genotype in the Japanese cohort [hazard ratio (HR): 0.43, P=0.001, and HR: 0.47, P=0.006, respectively]; this remained significant upon multivariable analysis adjusted for age, sex, stage, and type of adjuvant therapy (HR: 0.48; P=0.009, HR: 0.50; P=0.017, respectively). However, there was no significant association of the polymorphism with clinical outcome in the US and Austrian cohorts. When stratified by sex in the Japanese cohort, male individuals, but not female individuals, with the G allele maintained a clinical outcome benefit in both univariable and multivariable analyses. CONCLUSION: MET rs40239 may serve as a prognostic biomarker in locoregional gastric cancer. These data also suggest that genetic variants of c-MET may show sex-related differences in the impact on clinical outcome.

Plastin polymorphisms predict gender- and stage-specific colon cancer recurrence after adjuvant chemotherapy.

Tumor recurrence after curative resection remains a major problem in patients with locally advanced colorectal cancer treated with adjuvant chemotherapy. Genetic single-nucleotide polymorphisms (SNP) may serve as useful molecular markers to predict clinical outcomes in these patients and identify targets for future drug development. Recent in vitro and in vivo studies have demonstrated that the plastin genes PLS3 and LCP1 are overexpressed in colon cancer cells and play an important role in tumor cell invasion, adhesion, and migration. Hence, we hypothesized that functional genetic variations of plastin may have direct effects on the progression and prognosis of locally advanced colorectal cancer. We tested whether functional tagging polymorphisms of PLS3 and LCP1 predict time to tumor recurrence (TTR) in 732 patients (training set, 234; validation set, 498) with stage II/III colorectal cancer. The PLS3 rs11342 and LCP1 rs4941543 polymorphisms were associated with a significantly increased risk for recurrence in the training set. PLS3 rs6643869 showed a consistent association with TTR in the training and validation set, when stratified by gender and tumor location. Female patients with the PLS3 rs6643869 AA genotype had the shortest median TTR compared with those with any G allele in the training set [1.7 vs. 9.4 years; HR, 2.84; 95% confidence interval (CI), 1.32-6.1; P = 0.005] and validation set (3.3 vs. 13.7 years; HR, 2.07; 95% CI, 1.09-3.91; P = 0.021). Our findings suggest that several SNPs of the PLS3 and LCP1 genes could serve as gender- and/or stage-specific molecular predictors of tumor recurrence in stage II/III patients with colorectal cancer as well as potential therapeutic targets.