Aberrant Gene Expression Patterns Research Articles

Methylation of the Constitutive Androstane Receptor Is Involved in the Suppression of CYP2C19 in Hepatitis B Virus-Associated Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), one of the most dangerous malignancies with an increasing incidence and a high mortality rate, represents a major international health problem. HCC progression is known to involve genome-wide alteration of epigenetic modifications, leading to aberrant gene expression patterns. The activity of CYP2C19, an important member of the cytochrome P450 superfamily, was reported to be compromised in HCC, but the underlying mechanism remains unclear. To understand whether epigenetic modification in HCC is associated with a change in CYP2C19 activity, we evaluated the expression levels of CYP2C19 and its transcription factors by quantitative real-time polymerase chain reaction using mRNA extracted from both primary hepatocytes and paired tumor versus nontumor liver tissues of patients infected with hepatitis B virus (HBV). DNA methylation was examined by bisulfite sequencing and methylation-specific polymerase chain reaction. Our results indicated that CYP2C19 could be regulated by e-box methylation of the constitutive androstane receptor (CAR). Decreased CYP2C19 expression in tumorous tissues of HBV-infected patients with HCC was highly correlated with suppressed expression and promoter hypermethylation of CAR. Our study demonstrates that aberrant CAR methylation is involved in CYP2C19 regulation in HBV-related HCC and may play a role in liver tumorigenesis.

The promise of epigenomic therapeutics in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is often viewed to arise primarily by genetic alterations. However, today we know that many aspects of the cancer phenotype require a crosstalk among these genetic alterations with epigenetic changes. Indeed, aberrant gene expression patterns, driven by epigenetics are fixed by altered signaling from mutated oncogenes and tumor suppressors to define the PDAC phenotype. This conceptual framework may have significant mechanistic value and could offer novel possibilities for treating patients affected with PDAC. In fact, extensive investigations are leading to the development of small molecule drugs that reversibly modify the epigenome. These new ‘epigenetic therapeutics’ discussed herein are promising to fuel a new era of studies, by providing the medical community with new tools to treat this dismal disease.

RNA-seq analysis of hippocampal tissues reveals novel candidate genes for drug refractory epilepsy in patients with MTLE-HS

Array-based profiling studies have shown implication of aberrant gene expression patterns in epileptogenesis. We have performed transcriptome analysis of hippocampal tissues resected from patients with MTLE-HS using RNAseq approach. Healthy tissues from tumour margins obtained during tumour surgeries were used as non-epileptic controls. RNA sequencing was performed using standard protocols on Illumina HiSeq 2500 platform. Differential gene expression analysis of the RNAseq data revealed 56 significantly regulated genes in MTLE patients. Gene cluster analysis identified 3 important hubs of genes mostly linked to, neuroinflammation and innate immunity, synaptic transmission and neuronal network modulation which are supportive of intrinsic severity hypothesis of pharmacoresistance. This study identified various genes like FN1 which is central in our analysis, NEUROD6, RELN, TGFβR2, NLRP1, SCRT1, CSNK2B, SCN1B, CABP1, KIF5A and antisense RNAs like AQP4-AS1 and KIRREL3-AS2 providing important insight into the understanding of the pathophysiology or genomic basis of drug refractory epilepsy due to MTS.

Establishment of In Vitro FUS-Associated Familial Amyotrophic Lateral Sclerosis Model Using Human Induced Pluripotent Stem Cells.

SummaryAmyotrophic lateral sclerosis (ALS) is a late-onset motor neuron disorder. Although its neuropathology is well understood, the cellular and molecular mechanisms are yet to be elucidated due to limitations in the currently available human genetic data. In this study, we generated induced pluripotent stem cells (iPSC) from two familial ALS (FALS) patients with a missense mutation in the fused-in sarcoma (FUS) gene carrying the heterozygous FUS H517D mutation, and isogenic iPSCs with the homozygous FUS H517D mutation by genome editing technology. These cell-derived motor neurons mimicked several neurodegenerative phenotypes including mis-localization of FUS into cytosolic and stress granules under stress conditions, and cellular vulnerability. Moreover, exon array analysis using motor neuron precursor cells (MPCs) combined with CLIP-seq datasets revealed aberrant gene expression and/or splicing pattern in FALS MPCs. These results suggest that iPSC-derived motor neurons are a useful tool for analyzing the pathogenesis of human motor neuron disorders.

Retroviral Vectors for Cancer Gene Therapy.

Advances in molecular technologies have led to the discovery of many disease-related genetic mutations as well as elucidation of aberrant gene and protein expression patterns in several human diseases, including cancer. This information has driven the development of novel therapeutic strategies, such as the utilization of small molecules to target specific cellular pathways and the use of retroviral vectors to retarget immune cells to recognize and eliminate tumor cells. Retroviral-mediated gene transfer has allowed efficient production of T cells engineered with chimeric antigen receptors (CARs), which have demonstrated marked success in the treatment of hematological malignancies. As a safety point, these modified cells can be outfitted with suicide genes. Customized gene editing tools, such as clustered regularly interspaced short palindromic repeats-CRISPR-associated nucleases (CRISPR-Cas9), zinc-finger nucleases (ZFNs), or TAL-effector nucleases (TALENs), may also be combined with retroviral delivery to specifically delete oncogenes, inactivate oncogenic signaling pathways, or deliver wild-type genes. Additionally, the feasibility of retroviral gene transfer strategies to protect the hematopoietic stem cells (HSC) from the dose-limiting toxic effects of chemotherapy and radiotherapy was demonstrated. While some of these approaches have yet to be translated into clinical application, the potential implications for improved cellular replacement therapies to enhance and/or support the current treatment modalities are enormous.

Genome-Wide DNA Methylation Patterns of Bovine Blastocysts Developed In Vivo from Embryos Completed Different Stages of Development In Vitro

Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP). Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO) using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and chromosome segregation in IVP blastocyst groups. Furthermore, 1.6, 3.4, 3.9 and 9.4% of the differentially methylated regions that were overlapped to the transcriptome profile data were negatively correlated with the gene expression patterns in ZY, 4C, 16C and IVP blastocyst groups, respectively. Therefore, this finding indicated that suboptimal culture condition during preimplantation embryo development induced changes in the DNA methylation landscape of the resulting blastocysts in a stage dependent manner and the altered DNA methylation pattern was only partly explained the observed aberrant gene expression patterns of the blastocysts.

WITHDRAWN: Could some epigenetic modifications hold the key to opposing the metabolic syndrome?

// Giovanni Tarantino 1,2,* , Matteo Nicola Di Minno 1,* and Carmine Finelli 3,* 1 Department of Clinical Medicine and Surgery, Federico II University Medical School of Naples, Via Sergio Pansini, Naples, Italy 2 Centro Ricerche Oncologiche di Mercogliano, Istituto Nazionale Per Lo Studio E La Cura Dei Tumori “Fondazione Giovanni Pascale”, IRCCS, Italy, Italy 3 Department of Emergency and Internal Medicine, Ospedale S. Maria della Pieta – ASL Napoli 3 Sud, Nola (Na), Italy * These authors have contributed equally to this work Correspondence to: Giovanni Tarantino, email: // Keywords : non alcoholic fatty liver diseases, metabolic syndrome, epigenetics, microRNAs, PUFAs Received : January 27, 2016 Accepted : December 12, 2016 Epub : December 21, 2016 Abstract The role of epigenetic mechanisms in the pathogenesis of diseases has been increasingly recognized over recent years. Due to the intrinsic flexibility of the chromatin structure both in response to diet and environment, and as a result of foreseeable therapeutic interventions, epigenetics has emerged as a promising conceptual frame to investigate chronic degenerative disorders. This applies particularly to diet-driven and environment-driven metabolic disorders, such as obesity and its complications, including atherosclerosis and non-alcoholic fatty liver diseases (NAFLD). Current evidence strongly suggests that chronic inflammation is a landmark of obesity, atherosclerosis, and NAFLD, and that markers of inflammation are important predictors of cardiovascular risk factors. A crucial question is how pro-inflammatory gene expression patterns are established and maintained in the aforementioned illnesses. Epigenetic gene regulation is a partially new entry in the field of cardiovascular diseases. Epigenetic modifications provide attractive candidate disease mechanisms by explaining, in principle, how diet, environment and lifestyle might impose aberrant gene expression patterns in the individual’s lifetime, which can also be considered the result of trans-generational epigenetic inheritance. The main long-term goals in this field are to identify and understand the role of the epigenetic marks that could be used as early predictors of metabolic risk, and possibly develop drugs or diet-related treatments able to delay these epigenetic changes or even reverse them. Therefore, a continued and greater understanding of these mechanisms will eventually help identify individuals at high risk of the Metabolic Syndrome, and develop therapeutic interventions, in accordance with current global government strategy, utilizing also PUFA rich foods.

Granulin, a novel STAT3-interacting protein, enhances STAT3 transcriptional function and correlates with poorer prognosis in breast cancer.

Since the neoplastic phenotype of a cell is largely driven by aberrant gene expression patterns, increasing attention has been focused on transcription factors that regulate critical mediators of tumorigenesis such as signal transducer and activator of transcription 3 (STAT3). As proteins that interact with STAT3 may be key in addressing how STAT3 contributes to cancer pathogenesis, we took a proteomics approach to identify novel STAT3-interacting proteins. We performed mass spectrometry-based profiling of STAT3-containing complexes from breast cancer cells that have constitutively active STAT3 and are dependent on STAT3 function for survival. We identified granulin (GRN) as a novel STAT3-interacting protein that was necessary for both constitutive and maximal leukemia inhibitory factor (LIF)induced STAT3 transcriptional activity. GRN enhanced STAT3 DNA binding and also increased the time-integrated amount of LIF-induced STAT3 activation in breast cancer cells. Furthermore, silencing GRN neutralized STAT3-mediated tumorigenic phenotypes including viability, clonogenesis, and migratory capacity. In primary breast cancer samples, GRN mRNA levels were positively correlated with STAT3 gene expression signatures and with reduced patient survival. These studies identify GRN as a functionally important STAT3-interacting protein that may serve as an important prognostic biomarker and potential therapeutic target in breast cancer.

Abnormal gene expression profile reveals the common key signatures associated with clear cell renal cell carcinoma: a meta-analysis.

The aims of this study were to identify the common gene signatures of clear cell renal cell carcinoma (CCRCC), and to expand the respective protein-protein interaction networks associated with CCRCC regulation. For the latter, we utilized multiple gene expression data sets from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO), with which we could analyze the aberrant gene expression patterns at the transcriptome level that distinguish cancer from normal samples. We obtained the GSE781 and GSE6344 clear cell renal cell carcinoma gene expression datasets from GEO, which contained a total of 37 cancer and 37 normal samples. Subsequent R language analysis allowed identification of the differentially expressed genes. The genes that exhibited significant up or downregulation in cancers were entered into the Database for Annotation, Visualization, and Integrated Discovery to perform analysis of gene functional annotations, resulting in the generation of two protein-protein interaction networks that included the most significantly up or downregulated genes in CCRCC. These allowed us to identify the key factor genes, which could potentially be utilized to separate cancer versus normal samples. The differentially regulated genes are also highly likely to be functionally important regulatory factors in renal cell carcinoma: cell functions showing enrichment of these genes include amine biosynthetic and vitamin metabolic processes, ion binding, extracellular transport function, and regulation of biosynthesis. Together, the results from our study offer further reason to pursue diagnosis and therapy of CCRCC at the molecular level.

PRMT1 Activates Leukemic Stem Cell Program in MLL-Rearranged Leukemia

In MLL-rearranged leukemia, the Mixed Lineage Leukemia (MLL) gene undergoes chromosomal translocation that results in the loss of C-terminal histone methyltransferase SET domain, whereas the N-terminal of MLL gene fuses in-frame with one of the 60 identified partner genes. The resultant MLL fusion proteins lead to a characteristic aberrant gene expression pattern in human acute myeloid and lymphoblastic leukemia. Epigenetic dysregulation mediated by MLL fusion proteins has been suggested to be a key event in MLL-rearranged leukemia. It has been demonstrated that MLL-EEN/PRMT1 oncogenic complex induces transformation of primary myeloid progenitors via introduction of aberrant H4R3me2 at target Hoxloci. PRMT1 is the predominant protein arginine methyltransferase in mammals and is responsible for over 85% of arginine methylation activity in mammalian cells. Dysregulation of PRMT1 has been implicated in different cancers such as leukemia, suggesting the expression of PRMT1 is positively correlated with cancer progression and clinical parameters. Nevertheless, the leukemogenic role of PRMT1 in the establishment of leukemic stem cell (LSC) remains unclear.Previously we have demonstrated that a MLL fusion protein, MLL-EEN, can strongly enhance the self-renewal ability of murine primary hematopoietic cells through multiple rounds of replating assays. We have created a conditional Mll-Een invertor mouse model (MllEen/+) in which the expression of fusion protein is restricted to hematopoietic progenitors. Immunophenotypic analysis demonstrated a significant increase in the immature myeloid cell population (c-kit+Mac-1+) in bone marrow of MllEen/+ mice, suggesting that the expression of Mll-Een induces the development of acute myeloid leukemia. We have also established an Mll-Een expressing cell line from the bone marrow of MllEen/+ mouse. These leukemic cells can persistently form colonies and they also demonstrated deregulation of Hox genes, which is frequently observed in human leukemia cases. The leukemogenicity of Mll-Een is closely associated with Prmt1, which was demonstrated through knockdown of Prmt1. Strikingly, we discovered a subpopulation of CD41+Mll-Een expressing cells, which showed enhanced self-renewal ability in the serial colony forming assays. The percentage of CD41+ leukemic cells is reduced once Prmt1 was knocked down, suggesting that Prmt1 is crucial in the maintenance of this subpopulation of cells. In addition, the CD41+ cells showed enhanced expression of genes associated with hematopoietic stem cell (HSC) activities (Bmi-1, Runx1, Tal-1 and Lmo2), implying that part of the HSC transcriptional program has been re-activated in these cells. We therefore speculate that the CD41+ cells may represent a group of MLL leukemic cells that harbors strong stem cell features, and presumably functions as LSCs. The CD41+ leukemic cells will be further characterized by their LSC functions and CD41 can potentially serve as a novel LSC marker in MLL-rearranged leukemia. Taken together, studies on the role of PRMT1 can provide novel insights on the establishment of LSC and the development of effective clinical treatment for MLL-rearranged leukemia. DisclosuresNo relevant conflicts of interest to declare.

331 ABNORMAL DNA METHYLATION PATTERNS AND ALLELE-SPECIFIC EXPRESSION OF IMPRINTED GENES IN BOVINE-INDUCED PLURIPOTENT STEM CELLS

Pluripotency reacquisition of somatic cells has been achieved through nuclear transfer (NT) to oocytes and, more recently, through induction with pluripotency-related factors (iPS cells). However, the epigenetic reprogramming process that enables the derivation of both NT-derived cloned animals and iPS cells is usually incomplete, leading to unhealthy offspring and poorly reprogrammed iPS cell lines. These unfavourable outcomes result in part from abnormal genome DNA methylation that leads to aberrant gene expression patterns. For instance, differentially methylated regions (DMR) and monoalleleic expression of imprinted genes, essential for normal cellular commitment and early development, are thought to be severely disturbed by reprogramming techniques. Indeed, H19 and SNRPN, imprinted genes, were disturbed in bovine NT-derived embryos and fetuses. Herein we investigated whether the DMR and parent-of-origin expression of the imprinted genes H19 and SNRPN are also perturbed in iPS lines. To analyse the DMR methylation patterns and allelic expression of H19 and SNRPN using parental-specific polymorphisms, we derived multiple clones of bovine iPS (biPS) cells from an interspecies (Bos indicus × Bos taurus) fetal fibroblast (bFF) using transduction with a policystronic lentivirus containing mouse Oct4, Sox2 c-Myc, and Klf-4 transcription factors. The DNA methylation patterns were evaluated by bisulfite sequencing and allelic expression by designing allele-specific PCR probes. We also quantified transcript expression by RT-PCR of H19, IGF2, SNRPN, OCT4, and NANOG by normalization with 3 housekeeping genes (GAPDH, NAT1, and ACTB). The biPS lines were characterised by a high nuclear : cytoplasmic ratio, dome-shaped colonies, positive AP activity, embryoid body formation, in vitro and in vivo (teratoma) formation, and expression of pluripotency-related genes. Compared to the bFF cells, methylation analyses of H19 showed partial hypomethylation of the paternal DMR on 1 iPS cell line and partial demethylation of the CTCF-binding region in the DMR of 2 other biPS lines, indicating abnormal demethylation of 3 out of the 4 biPS lines analysed. Methylation analyses of SNRPN revealed a partial hypomethylation in the maternal DMR and partial hypermethylation of the paternal DMR in 2 iPS lines. Gene expression analyses revealed the biallelic expression of H19 and decreased global expression of both H19 and IGF2, as well as the exclusively monoallelic paternal expression and significant increase in global expression of SNRPN. Interestingly, although OCT4 was substantially overexpressed in biPS lines, we identified a hypermethylation of the CG-rich region of the OCT4 exon 1. Endogenous NANOG expression was observed in 2 biPS clones. We conclude that imprinting errors are observed in biPS clones, suggesting that these epigenetic anomalies are related to the reprogramming process and could be directly responsible for the variable phenotypes and low success rates of both cloning and iPS derivation procedures.Financial support was from NSERC, FAPESP (13/13686-8, 11/08376-4, 57877-3/2008, 08.135-2/2013), CNPq (573754/2008-0, 482163/2013-5).

OP1-05 INTEGRATIVE DNA METHYLATION AND GENE EXPRESSION ANALYSES IDENTIFIES DISCOIDIN DOMAIN RECEPTOR 1 (DDR1) ASSOCIATION WITH IDIOPATHIC NONOBSTRUCTIVE AZOOSPERMIA

You have accessJournal of UrologyOutstanding Posters: Research1 Apr 2014OP1-05 INTEGRATIVE DNA METHYLATION AND GENE EXPRESSION ANALYSES IDENTIFIES DISCOIDIN DOMAIN RECEPTOR 1 (DDR1) ASSOCIATION WITH IDIOPATHIC NONOBSTRUCTIVE AZOOSPERMIA Ranjith Ramasamy, Alex Ridgeway, Josephine Addai, Larry Lipshultz, and Dolores Lamb Ranjith RamasamyRanjith Ramasamy More articles by this author , Alex RidgewayAlex Ridgeway More articles by this author , Josephine AddaiJosephine Addai More articles by this author , Larry LipshultzLarry Lipshultz More articles by this author , and Dolores LambDolores Lamb More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.613AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Spermatogenesis is a complex process that involves proliferation, differentiation, and cell adhesion. Spermatogenic failure or non−obstructive azoospermia (NOA) results from mechanisms involved are incompletely understood. DDR1 is a member of a small subfamily of receptor tyrosine kinases that is involved in adhesion, migration, proliferation, apoptosis, cell morphogenesis and differentiation. Since, DDR1 is expressed in human post−meiotic germ cells of testis, we hypothesized that abnormal DDR1 expression could be a possible mechanism that can compromise spermatogenesis in a subset of men with idiopathic NOA. METHODS We used the high resolution Infinium 450K methylation array and compared fibrobalsts cultured from testicular biopsies of 19 NOA men and 4 fertile controls. Microarray data was analyzed using Minfi (R software package) utilizing subset−quantile within array normalization. We investigated the functional role of abnormal promoter DNA methylation for selected genes using mRNA expression by quantitative RT−real time PCR. Immunohistochemistry was used to confirm testicular expression and potential importance in spermatogenesis RESULTS Differentially methylated CpG sites (∼20K) were identified using an F−Test (p<0.05) in the NOA samples. We identified 24 genes with the >30% difference in methylation within promoter region of men with NOA and fertile controls. Of the aberrantly methylated CpGs, 13 were hypomethylated and 11 were hypermethylated groups. From the top 11 hypermethylated genes, six genes (MRI1, DCAF12L1, TMEM95, CECR2, DDR1, NPHS2) were selected for validation since they were shown to be expressed in testis. Of the 6 genes validated with qPCR, DDR1 showed aberrant gene expression pattern. Four (21%) patients out of the 19 NOA men had lower expression levels (1.8x) of DDR1, whereas two (10.5%) men had higher expression levels (2.5x) of DDR1compared to fertile men (p<0.05). Immunohistochemical analysis suggests presence of DDR1 within cytoplasm of germ cells in fertile men and men with maturation arrest histology. DDR1 protein is absent in men with Sertoli−cell only or germ cell aplasia. CONCLUSIONS Aberrant expression of DDR1 is associated with NOA. The functional relevance of abnormal methylation of DDR1 to NOA warrants further investigation © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e163-e164 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Ranjith Ramasamy More articles by this author Alex Ridgeway More articles by this author Josephine Addai More articles by this author Larry Lipshultz More articles by this author Dolores Lamb More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

EMT and Acquisition of Stem Cell-Like Properties Are Involved in Spontaneous Formation of Tumorigenic Hybrids between Lung Cancer and Bone Marrow-Derived Mesenchymal Stem Cells

The most deadly phase in cancer progression is metastatic conversion. Epithelial-to-mesenchymal transition (EMT) is a key process by which cancer cells acquire invasive and metastatic phenotypes. In order to spawn macroscopic metastases, disseminated cancer cells would seem to require self-renewal capability. However, the underlying mechanism defining these processes is poorly understood. One possible mechanism underlying metastasis is fusion between myeloid cells and cancer cells. In this study, we found that spontaneously-formed tumorigenic hybrids between bone marrow-derived mesenchymal stem cells (MSCs) and three different non-small cell lung cancer (NSCLC) cell lines contributed to highly malignant subpopulations with both EMT and stem cell-like properties. Hybrids lost their epithelial morphology and assumed a fibroblast-like appearance. Up-regulation of vimentin, α-smooth muscle actin (α-SMA), and fibronectin, and down-regulation of E-cadherin and pancytokeratin were observed in tumorigenic hybrids. These cells also exhibited increased expression of the stem cell marker prominin-1 (CD133) and over-expression of transcription factors OCT4, Nanog, BMI1, Notch1, ALDH1 as well as Sox2, all genes responsible for regulating and maintaining the stem cell phenotype. In addition, in spontaneously-formed tumorigenic hybrids, increased pneumosphere-forming capacity and tumor-forming ability in NOD/SCID mice were detectable. Thus, cell fusion between lung cancer cells and MSCs provides a nonmutational mechanism that could contribute to aberrant gene expression patterns and give rise to highly malignant subpopulations both capable of EMT and with properties of cancer stem cells (CSCs).

Targeting transcription factors

A lack of effective treatments for advanced cancer remains a major challenge in oncology. Because cancer is a disease associated with aberrant gene expression patterns, transcription factors, which serve as the convergence points of oncogenic signaling and are functionally altered in many cancers, hold great therapeutic promise. Many human cancers are dependent on the inappropriate activity of oncogenic transcription factors. By contrast, normal cells can often tolerate disruption of these proteins with little toxicity. Direct inhibition of transcription factor expression (e.g., with RNA interference or microRNAs) and DNA binding (e.g., with oligodeoxynucleotide decoys or pyrrole-imidazole polyamides) has demonstrated antitumor responses with minimal side-effects. New strategies of targeting transcription factors include disrupting critical protein-protein interactions, and restricting binding at the epigenetic level by modulating chromatin accessibility. Moreover, targeting transcription factors in tumor-associated immune cells has the potential to overcome tumor immunoresistance. Transcription factors are an important target for cancer therapy, both through direct anticancer effects and immunomodulatory actions. Newly developed delivery systems that specifically target tumor cells also create opportunities for successes in targeting transcription in cancer.

Hepatic leukemia factor promotes resistance to cell death: Implications for therapeutics and chronotherapy

Physiological variation related to circadian rhythms and aberrant gene expression patterns are believed to modulate therapeutic efficacy, but the precise molecular determinants remain unclear. Here we examine the regulation of cell death by hepatic leukemia factor (HLF), which is an output regulator of circadian rhythms and is aberrantly expressed in human cancers, using an ectopic expression strategy in JB6 mouse epidermal cells and human keratinocytes. Ectopic HLF expression inhibited cell death in both JB6 cells and human keratinocytes, as induced by serum-starvation, tumor necrosis factor alpha and ionizing radiation. Microarray analysis indicates that HLF regulates a complex multi-gene transcriptional program encompassing upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes, and many additional changes that are consistent with an anti-death program. Collectively, our results demonstrate that ectopic expression of HLF, an established transcription factor that cycles with circadian rhythms, can recapitulate many features associated with circadian-dependent physiological variation.

Role of the hypothalamic–pituitary–adrenal axis in developmental programming of health and disease

Adverse environments during the fetal and neonatal development period may permanently program physiology and metabolism, and lead to increased risk of diseases in later life. Programming of the hypothalamic-pituitary-adrenal (HPA) axis is one of the key mechanisms that contribute to altered metabolism and response to stress. Programming of the HPA axis often involves epigenetic modification of the glucocorticoid receptor (GR) gene promoter, which influences tissue-specific GR expression patterns and response to stimuli. This review summarizes the current state of research on the HPA axis and programming of health and disease in the adult, focusing on the epigenetic regulation of GR gene expression patterns in response to fetal and neonatal stress. Aberrant GR gene expression patterns in the developing brain may have a significant negative impact on protection of the immature brain against hypoxic-ischemic encephalopathy in the critical period of development during and immediately after birth.

Aberrant gene expression patterns in extraembryonic tissue from cloned porcine embryos

The abnormal development of embryos reconstructed by somatic cell nuclear transfer (SCNT) is considered to be associated with consequent changes in gene expression following errors in epigenetic reprogramming. In this study, we carried out SCNT using donor fibroblast cells derived from 3-way hybrids (Landrace×Duroc×Yorkshire). A total of 655 SCNT embryos were transferred, and 6.97±2.3 cloned fetuses were successfully recovered from three surrogates at gestational day 30. An analysis of the 6.97±2.3 cloned embryos revealed that most had severe extraembryonic defects. The extraembryonic tissue from the SCNT embryos was abnormally small compared with that of the control. To investigate the differentially expressed genes between the SCNT and control extraembryonic tissues, we compared the gene expression profiles of the extraembryonic tissues from gestational day 30 cloned pig embryos with those from the control using an annealing control primer-based GeneFishing polymerase chain reaction. As a result, we found that a total of 50 genes were differentially expressed by utilizing 120 ACPs, 38 genes of which were known. Among them, 26 genes were up-regulated, whereas 12 genes were down-regulated. Real-time RT-PCR showed that apoptosis-related genes were expressed significantly higher in SCNT extraembryonic tissue than in the control, whereas metabolism-related genes were expressed at significantly lower levels in the SCNT extraembryonic tissue. These observations strongly indicate that early gestational death of SCNT embryo is caused, at least in part, by the disruption of developing extraembryonic tissues as a result of aberrant gene expression, which results in abnormal apoptosis and metabolism.

Aberrant placenta gene expression pattern in bovine pregnancies established after transfer of cloned or in vitro produced embryos

In the present study, we used the global transcriptome profile approach to identify dysregulated genes, molecular pathways, and molecular functional alterations in bovine placentas derived from somatic cell nuclear transfer (SCNT) and in vitro embryo production (IVP) pregnancies compared with their artificial insemination (AI) counterparts at day 50 of gestation. For this, day 7 blastocysts derived from AI, IVP, or SCNT were transferred to oestrus-synchronized cows. The pregnant animals were slaughtered at day 50 of gestation, and the placentas were then recovered and used for transcriptome analysis using Affymetrix GeneChip bovine genome array. Results showed the SCNT placenta to be different from its AI counterpart in the expression of 1,196 transcripts. These genes were found to be associated with alterations in key biological processes and molecular pathways in SCNT placenta, and the dysregulation of 9% (n = 110) of these genes was due to transcriptional reprogramming error. IVP placenta also displayed alterations in the expression of 72 genes, of which 58 were common to SCNT placenta. Gene enrichment analysis revealed that the expression of genes involved in organ development, blood vessel development, extracellular matrix organization, and the immune system was affected in both SCNT and IVP placentas. However, 96% of the affected genes in SCNT were not significantly altered in IVP groups. Thus, the higher transcriptome dysregulation in SCNT placenta followed by IVP would reflect the degree of placental abnormality in SCNT and IVP pregnancies at day 50 of the gestation, which may have a profound effect on subsequent fetal development and health of the offspring.

Disrupted Redox Homeostasis and Aberrant Redox Gene Expression in Porcine Oocytes Contribute to Decreased Developmental Competence1

The objective of this study was to identify specific redox-related genes whose function contributes to oocyte quality and to characterize the role of redox homeostasis in oocyte development. We determined the redox genes glutaredoxin 2 (GLRX2), protein disulfide isomerase family A, members 4 and 6 (PDIA4, PDIA6), and thioredoxin reductase 1 (TXNRD1) were differentially expressed between adult (more competent) and prepubertal (less competent) porcine in vitro-matured (IVM) oocytes. The association between these genes and oocyte quality was further validated by comparing transcript abundance in IVM with that in in vivo-matured (VVM) prepubertal and adult oocytes. By maturing oocytes in variable redox environments, we demonstrated that a balanced redox environment is important for oocyte quality, and over-reduction of the environment is as detrimental as excess oxidation. Critical levels of reactive oxygen species (ROS) and glutathione (GSH) are required for oocyte competence. Elevated GSH and lower ROS in prepubertal oocytes suggest disrupted redox homeostasis exists in these cells. By further comparing GLRX2, PDIA4, PDIA6, and TXNRD1 expression levels in oocytes matured under these different redox environments, we found aberrant expression patterns in prepubertal oocytes but not in adult oocytes when the maturation medium contained high concentrations of antioxidants. These results suggest that prepubertal oocytes are less competent in regulating redox balance than adult oocytes, contributing to lower oocyte quality. In conclusion, aberrant redox gene expression patterns and disrupted redox homeostasis contribute to decreased developmental competence in prepubertal and IVM porcine oocytes. The balance between ROS and GSH plays an important role in oocyte quality.

Altered Cell Cycle Gene Expression and Apoptosis in Post-Implantation Dog Parthenotes

Mature oocytes can be parthenogenetically activated by a variety of methods and the resulting embryos are valuable for studies of the respective roles of paternal and maternal genomes in early mammalian development. In the present study, we report the first successful development of parthenogenetic canine embryos to the post-implantation stage. Nine out of ten embryo transfer recipients became pregnant and successful in utero development of canine parthenotes was confirmed. For further evaluation of these parthenotes, their fetal development was compared with artificially inseminated controls and differentially expressed genes (DEGs) were compared using ACP RT-PCR, histological analysis and immunohistochemistry. We found formation of the limb-bud and no obvious differences in histological appearance of the canine parthenote recovered before degeneration occurred; however canine parthenotes were developmentally delayed with different cell cycle regulating-, mitochondria-related and apoptosis-related gene expression patterns compared with controls. In conclusion, our protocols were suitable for activating canine oocytes artificially and supported early fetal development. We demonstrated that the developmental abnormalities in canine parthenotes may result from defective regulation of apoptosis and aberrant gene expression patterns, and provided evidence that canine parthenotes can be a useful tool for screening and for comparative studies of imprinted genes.