Localized prostate cancer (PCa) is often curable, whereas metastatic disease treated by castration inevitably progresses toward castration-resistant PCa (CRPC). Most CRPC treatments target androgen receptor (AR) signaling. However, not all CRPC cells rely on AR activity for survival and proliferation. With advances in immunotherapy and fluid biopsies for cancer management, expression systems specific for both AR-positive and -negative PCa are required for virus-based vaccines and cell imaging. To target both AR-responsive and non-responsive cells, we developed a three-step transcriptional amplification (3STA) system based on the progression elevated gene-3 (

AIM: To investigate the regulatory potential of intergenic/intronic hypomethylated regions (iHMRs) within imprinted domains.
MATERIALS & METHODS: Based on the preliminary results of the histone modification and conservation profiles, we conducted reporter assays on the Peg3 and H19 domain iHMRs. The in vitro results were confirmed by the in vivo deletion of Peg3-iHMR designed to test its function in the Peg3 imprinted domain.
RESULTS & CONCLUSION: Initial bioinformatic analyses suggested that some iHMRs may be noncanonical enhancers for imprinted genes. Consistent with this, Peg3- and H19-iHMRs showed context-dependent promoter and enhancer activity. Further, deletion of Peg3-iHMR resulted in allele- and sex-specific misregulation of several imprinted genes within the domain. Taken together, these results suggest that some iHMRs may function as domain-wide regulators for the associated imprinted domains.

Deregulation of microRNAs (miRNAs) contributes to the development and progression of many cancer types; however, their functions in the pathogenesis of testicular germ cell tumor (TGCT) remain unclear. Here, we determined miRNA expression profiles of TGCTs and normal testes using small RNA sequencing, and identified several deregulated miRNAs in TGCTs, including the miR-506~514 cluster. In functional studies in vitro we demonstrated that miR-514a-3p induced apoptosis through direct regulation of the paternally expressed gene 3 (PEG3), and ectopically expressed PEG3 could rescue the apoptotic effect of miR-514a-3p overexpression. Silencing of PEG3 or miR-514a-3p overexpression reduced nuclear accumulation of p50 and NF-κB reporter activity. Furthermore, PEG3 was co-immunoprecipitated with tumor necrosis factor receptor-associated factor 2 (TRAF2) in TGCT cell lysates. We propose a model of PEG3-mediated activation of NF-κB in TGCT. Loss of miR-514a-3p expression in TGCT increases PEG3 expression that recruits TRAF2 and activates the NF-kappa B pathway, which protects germ cells from apoptosis. Importantly, we observed strong expression of PEG3 and nuclear p50 in the majority of TGCTs (83% and 78%, respectively). In conclusion, our study describes a novel function for miR-514a-3p in TGCT and highlights an unrecognized mechanism of PEG3 regulation and NF-κB activation in TGCT.

The homogeneity and heterogeneity in somatic mutations, copy number alterations and methylation across different cancer types have been extensively explored. However, the related exploration based on transcriptome data is lacking. In this study we explored gene expression profiles across 33 human cancer types using The Cancer Genome Atlas (TCGA) data. We identified consistently upregulated genes (such as E2F1, EZH2, FOXM1, MYBL2, PLK1, TTK, AURKA/B and BUB1) and consistently downregulated genes (such as SCARA5, MYOM1, NKAPL, PEG3, USP2, SLC5A7 and HMGCLL1) across various cancers. The dysregulation of these genes is likely to be associated with poor clinical outcomes in cancer. The dysregulated pathways commonly in cancers include cell cycle, DNA replication, repair, and recombination, Notch signaling, p53 signaling, Wnt signaling, TGFβ signaling, immune response etc. We also identified genes consistently upregulated or downregulated in highly-advanced cancers compared to lowly-advanced cancers. The highly (low) expressed genes in highly-advanced cancers are likely to have higher (lower) expression levels in cancers than in normal tissue, indicating that common gene expression perturbations drive cancer initiation and cancer progression. In addition, we identified a substantial number of genes exclusively dysregulated in a single cancer type or inconsistently dysregulated in different cancer types, demonstrating the intertumor heterogeneity. More importantly, we found a number of genes commonly dysregulated in various cancers such as PLP1, MYOM1, NKAPL and USP2 which were investigated in few cancer related studies, and thus represent our novel findings. Our study provides comprehensive portraits of transcriptional landscape of human cancers.

Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2-8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer (

BACKGROUND: Genomic imprinting is associated with many human diseases, including various types of cancers, however, no studies on gene imprinting are related to squamous cell carcinoma of the head and neck (SCCHN) directly.
MATERIALS AND METHODS: In this study, the expression of a panel of 15 imprinted genes in cancerous and non-cancerous tissues from 73 patients with SCCHN were investigated.
RESULTS: Altered expression of carboxypeptidase A4 (CPA4); protein phosphatase 1 regulatory subunit 9A (PPP1R9A); H19, imprinted maternally expressed transcript (non-protein coding) (H19); paternally expressed gene 3 antisense RNA 1 (PEG3-AS1); retrotransposon-like 1 (RTL1), insulin-like growth factor 2 (IGF2); solute carrier family 22 member 3 (SLC22A3); and gamma-aminobutyric acid type A receptor beta3 subunit (GABRB3) was observed. Down-regulation of PPP1R9A (p<0.05) and GABRB3 (p<0.05) was correlated with more advanced cancer stages. Down-regulation of PEG3-AS1 (p<0.05) and GABRB3 (p<0.01) was correlated with lymph node metastasis. Poor survival was related to higher expression of CPA4 (p<0.01) and lower expression of PEG3-AS1 (p<0.05) and IGF2 (p<0.05). Chemotherapy was also found to have an impact on the expression of imprinted genes.
CONCLUSION: Loss of imprinting is involved in tumorigenesis of SCCHN.

AIM: This study aimed to clarify the genetic and epigenetic features of recurrent hydatidiform mole (RHM) in Japanese patients.
METHODS: Four Japanese isolated RHM cases were analyzed using whole-exome sequencing. Villi from RHMs were collected by laser microdissection for genotyping and DNA methylation assay of differentially methylated regions (DMRs). Single nucleotide polymorphisms of PEG3 and H19 DMRs were used to confirm the parental origin of the variants.
RESULTS: A novel homozygous nonsense mutation in NLRP7 (c.584G>A; p.W195X) was identified in 1 patient. Genotyping of one of her molar tissue revealed that it was biparental but not androgenetic in origin. Despite the fact that the RHM is biparental, maternally methylated DMRs of PEG3, SNRPN and PEG10 showed complete loss of DNA methylation. A paternally methylated DMR of H19 retained normal methylation.
CONCLUSIONS: This is the first Japanese case of RHM with a novel homozygous nonsense NLRP7 mutation and a specific loss of maternal DNA methylation of DMRs. Notably, the mutation was identified in an isolated case of an ethnic background that has not previously been studied in this context. Our data underscore the involvement of NLRP7 in RHM pathophysiology and confirm that DNA methylation of specific regions is critical.

Decorin is a prototypical small leucine-rich proteoglycan that epitomizes the multifunctional nature of this critical gene family. Soluble decorin engages multiple receptor tyrosine kinases within the target-rich environment of the tumor stroma and tumor parenchyma. Upon receptor binding, decorin initiates signaling pathways within endothelial cells downstream of VEGFR2 that ultimately culminate in a Peg3/Beclin 1/LC3-dependent autophagic program. Concomitant with autophagic induction, decorin blunts capillary morphogenesis and endothelial cell migration, thereby significantly compromising tumor angiogenesis. In parallel within the tumor proper, decorin binds multiple RTKs with high affinity, including Met, for a multitude of oncosuppressive functions including growth inhibition, tumor cell mitophagy, and angiostasis. Decorin is also pro-inflammatory by modulating macrophage function and cytokine secretion. Decorin suppresses tumorigenic growth, angiogenesis, and prevents metastatic lesions in a variety of in vitro and in vivo tumor models. Therefore, decorin would be an ideal therapeutic candidate for combating solid malignancies.

Many imprinted genes are often epigenetically affected in human cancers due to their functional linkage to insulin and insulin-like growth factor signaling pathways. Thus, the current study systematically characterized the epigenetic instability of imprinted genes in multiple human cancers. First, the survey results from TCGA (The Cancer Genome Atlas) revealed that the expression levels of the majority of imprinted genes are downregulated in primary tumors compared to normal cells. These changes are also accompanied by DNA methylation level changes in several imprinted domains, such as the PEG3, MEST and GNAS domains. Second, these DNA methylation level changes were further confirmed manually using several sets of cancer DNA. According to the results, the Imprinting Control Regions of the PEG3, MEST and GNAS domains are indeed affected in breast, lung and ovarian cancers. This DNA methylation survey also revealed that evolutionarily conserved cis-regulatory elements within these imprinted domains are very variable in both normal and cancer cells. Overall, this study highlights the epigenetic instability of imprinted domains in human cancers and further suggests its potential use as cancer biomarkers.

PURPOSE OF REVIEW: We review the genetic, epigenetic and transcriptional landscape of liver fluke (Opisthorchis viverrini, Ov)-related cholangiocarcinoma (CCA). Its distinct alterations, as compared with non-Ov-related CCA may help shed light on its underlying molecular mechanisms.
RECENT FINDINGS: Recent whole-exome and targeted sequencing not only confirmed frequent mutations in known CCA-related genes including TP53 (44%), KRAS (16.7%) and SMAD4 (16.7%), but also revealed mutations in novel CCA-related genes associated with chromatin remodeling [BAP1 (2.8%), ARID1A (17.6%), MLL3 (13%) and IDH1/2 (2.8%)], WNT signaling [RNF43 (9.3%) and PEG3 (5.6%)] and KRAS/G protein signaling [GNAS (9.3%) and ROBO2 (9.3%)]. Interestingly, there is a significant difference in the frequency of mutated genes between Ov-related CCA and non-Ov-related CCA, such as p53 and IDH1/2, reflecting the impact of cause on pathogenesis. Altered DNA methylation and transcriptional profiles associated with xenobiotic metabolism and pro-inflammatory responses were also found in Ov-related CCA.
SUMMARY: Liver fluke-induced chronic inflammation plays a crucial role in cholangiocarcinogenesis, resulting in distinct signatures of genetic, epigenetic and transcriptional alterations. These alterations, when contrasted with non-Ov-related CCA, indicate a unique pathogenic process in Ov-related CCA and may have potential clinical implications on diagnostics, therapeutics and prevention.

INTRODUCTION: Real-time monitoring of biologic changes in tumors may be possible by investigating the transitional cells such as circulating tumor cells (CTCs) and disseminated tumor cells in bone marrow (BM-DTCs). However, the small numbers of CTCs and the limited access to bone marrow aspirates in cancer patients pose major hurdles. The goal of this study was to determine whether breast cancer (BC) patient-derived xenograft (PDX) mice could provide a constant and renewable source of CTCs and BM-DTCs, thereby representing a unique system for the study of metastatic processes.
METHODS: CTCs and BM-DTCs, isolated from BC PDX-bearing mice, were identified by immunostaining for human pan-cytokeratin and nuclear counterstaining of red blood cell-lysed blood and bone marrow fractions, respectively. The rate of lung metastases (LM) was previously reported in these lines. Associations between the presence of CTCs, BM-DTCs, and LM were assessed by the Fisher's Exact and Cochran-Mantel-Haenszel tests. Two separate genetic signatures associated with the presence of CTC clusters and with lung metastatic potential were computed by using the expression arrays of primary tumors from different PDX lines and subsequently overlapped to identify common genes.
RESULTS: In total, 18 BC PDX lines were evaluated. CTCs and BM-DTCs, present as either single cells or clusters, were detected in 83% (15 of 18) and 62.5% (10 to16) of the lines, respectively. A positive association was noted between the presence of CTCs and BM-DTCs within the same mice. LM was previously found in 9 of 18 (50%) lines, of which all nine had detectable CTCs. The presence of LM was strongly associated with the detection of CTC clusters but not with individual cells or detection of BM-DTCs. Overlapping of the two genetic signatures of the primary PDX tumors associated with the presence of CTC clusters and with lung metastatic potential identified four genes (HLA-DP1A, GJA1, PEG3, and XIST). This four-gene profile predicted distant metastases-free survival in publicly available datasets of early BC patients.
CONCLUSION: This study suggests that CTCs and BM-DTCs detected in BC PDX-bearing mice may represent a valuable and unique preclinical model for investigating the role of these rare cells in tumor metastases.

Epigenetic regulation of imprinted genes enables monoallelic expression according to parental origin, and its disruption is implicated in many cancers and developmental disorders. The expression of hormone receptors is significant in breast cancer because they are indicators of cancer cell growth rate and determine response to endocrine therapies. We investigated the frequency of aberrant events and variation in DNA methylation at nine imprinted sites in invasive breast cancer and examined the association with estrogen and progesterone receptor status. Breast tissue and blood from patients with invasive breast cancer (n = 38) and benign breast disease (n = 30) were compared with those from healthy individuals (n = 36), matched with the cancer patients by age at diagnosis, ethnicity, body mass index, menopausal status and familial history of cancer. DNA methylation and allele-specific expression were analyzed by pyrosequencing. Tumor-specific methylation changes at IGF2 DMR2 were observed in 59% of cancer patients, IGF2 DMR0 in 38%, DIRAS3 DMR in 36%, GRB10 ICR in 23%, PEG3 DMR in 21%, MEST ICR in 19%, H19 ICR in 18%, KvDMR in 8% and SNRPN/SNURF ICR in 4%. Variation in methylation was significantly greater in breast tissue from cancer patients compared with that in healthy individuals and benign breast disease. Aberrant methylation of three or more sites was significantly associated with negative estrogen-alpha (Fisher's exact test, p = 0.02) and progesterone-A (p = 0.02) receptor status. Aberrant events and increased variation in imprinted gene DNA methylation, therefore, seem to be frequent in invasive breast cancer and are associated with negative estrogen and progesterone receptor status, without loss of monoallelic expression.

PURPOSE: Tumor heterogeneity and subsistence of high-grade serous ovarian adenocarcinoma (HGSC) classes can be speculated from clinical incidences suggesting passive tumor dissemination versus active invasion and metastases.
EXPERIMENTAL DESIGN: We explored this theme toward tumor classification through two approaches of gene expression pattern clustering: (i) derivation of a core set of metastases-associated genes and (ii) resolution of independent weighted correlation networks. Further identification of appropriate cell and xenograft models was carried out for resolution of class-specific biologic functions.
RESULTS: Both clustering approaches achieved resolution of three distinct tumor classes, two of which validated in other datasets. Networks of enriched gene modules defined biologic functions of quiescence, cell division-differentiation-lineage commitment, immune evasion, and cross-talk with niche factors. Although deviant from normal homeostatic mechanisms, these class-specific profiles are not totally random. Preliminary validation of these suggests that Class 1 tumors survive, metastasize in an epithelial-mesenchymal transition (EMT)-independent manner, and are associated with a p53 signature, aberrant differentiation, DNA damage, and genetic instability. These features supported by association of cell-specific markers, including PAX8, PEG3, and TCF21, led to the speculation of their origin being the fimbrial fallopian tube epithelium. On the other hand, Class 2 tumors activate extracellular matrix-EMT-driven invasion programs (Slug, SPARC, FN1, THBS2 expression), IFN signaling, and immune evasion, which are prospectively suggestive of ovarian surface epithelium associated wound healing mechanisms. Further validation of these etiologies could define a new therapeutic framework for disease management.

BACKGROUND: Mesenchymal hamartoma of the liver (MHL) is an uncommon benign primary liver tumor that typically occurs in the pediatric population, although cases have been described in adults. MHL is sometimes associated with the highly malignant undifferentiated embryonal sarcoma (UES), and the synchronous or metachronous occurrence of MHL and UES suggests they share a common genetic link. Although the exact mechanism of tumorigenesis has not been identified, MHL cases harbor recurring chromosomal rearrangements involving 19q13.
DESIGN: In order to provide more details on the genetic events of MHL tumorigenesis, capture-based next generation sequencing (NGS) targeted to loci recently shown to be involved in a translocation in a case of UES arising in MHL (specifically, the MALAT1 gene on chromosome 11 and a gene poor region termed MHLB1 on chromosome 19) was performed on formalin fixed paraffin embedded tissue from seven cases of MHL.
RESULTS: Chromosome rearrangements involving the MHLB1 locus were identified in three of the seven cases, including the translocation t(11,19)(q13.1;q13.42) involving the MALAT1 gene; the translocation t(2,19)(q31.1;q13.42) involving AK023515, an uncharacterized noncoding gene; and the inversion inv(19,19)(q13.42;q13.43) involving the PEG3 gene encoding a Kruppel-type zinc-finger protein. Rearrangements were exclusively identified in pediatric tumors. In each case, the presence of the rearrangement was confirmed by PCR and interphase FISH. Interphase FISH also demonstrated that the arrangements occur within the spindle cell component but not within the epithelial components of the tumor.
CONCLUSIONS: Since the MHLB1 locus contains a CpG-rich region whose methylation regulates C19MC miRNA genes, rearrangements that disrupt this region may contribute to MHL development through alteration of miRNA expression. The demonstration that the loose stromal cells harbor the rearrangements indicates that (some cases of) MHL are a neoplastic process due to a somatic genetic change and not a germline abnormality.

OBJECTIVES: Low birth weight (LBW) has been associated with common adult-onset chronic diseases, including obesity, cardiovascular disease, type II diabetes and some cancers. The etiology of LBW is multi-factorial. However, recent evidence suggests exposure to antibiotics may also increase the risk of LBW. The mechanisms underlying this association are unknown, although epigenetic mechanisms are hypothesized. In this study, we evaluated the association between maternal antibiotic use and LBW and examined the potential role of altered DNA methylation that controls growth regulatory imprinted genes in these associations.
METHODS: Between 2009-2011, 397 pregnant women were enrolled and followed until delivery. Prenatal antibiotic use was ascertained through maternal self-report. Imprinted genes methylation levels were measured at differentially methylated regions (DMRs) using bisulfite pyrosequencing. Generalized linear models were used to examine associations among antibiotic use, birth weight and DMR methylation fractions.
RESULTS: After adjusting for infant gender, race/ethnicity, maternal body mass index, delivery route, gestational weight gain, gestational age at delivery, folic acid intake, physical activity, maternal smoking and parity, antibiotic use during pregnancy was associated with 138 g lower birth weight compared with non-antibiotic use (β-coefficient=-132.99, s.e.=50.70, P=0.008). These associations were strongest in newborns of women who reported antibiotic use other than penicillins (β-coefficient=-135.57, s.e.=57.38, P=0.02). Methylation at five DMRs, IGF2 (P=0.05), H19 (P=0.15), PLAGL1 (P=0.01), MEG3 (P=0.006) and PEG3 (P=0.08), was associated with maternal antibiotic use; among these, only methylation at the PLAGL1 DMR was also associated with birth weight.
CONCLUSION: We report an inverse association between in utero exposure to antibiotics and lower infant birth weight and provide the first empirical evidence supporting imprinted gene plasticity in these associations.

Cytology-based screening for invasive cervical cancer (ICC) lacks sensitivity and specificity to discriminate between cervical intraepithelial neoplasia (CIN) likely to persist or progress from cases likely to resolve. Genome-wide approaches have been used to identify DNA methylation marks associated with CIN persistence or progression. However, associations between DNA methylation marks and CIN or ICC remain weak and inconsistent. Between 2008-2009, we conducted a hospital-based, case-control study among 213 Tanzania women with CIN 1/2/3 or ICC. We collected questionnaire data, biopsies, peripheral blood, cervical scrapes, Human papillomavirus (HPV) and HIV-1 infection status. We assessed PEG3 methylation status by bisulfite pyrosequencing. Multinomial logistic regression was used to estimate odds ratios (OR) and confidence intervals (CI 95%) for associations between PEG3 methylation status and CIN or ICC. After adjusting for age, gravidity, hormonal contraceptive use and HPV infection, a 5% increase in PEG3 DNA methylation was associated with increased risk for ICC (OR = 1.6; 95% CI 1.2-2.1). HPV infection was associated with a higher risk of CIN1-3 (OR = 15.7; 95% CI 5.7-48.6) and ICC (OR = 29.5, 95% CI 6.3-38.4). Infection with high risk HPV was correlated with mean PEG3 differentially methylated regions (DMRs) methylation (r = 0.34 p<0.0001), while the correlation with low risk HPV infection was weaker (r = 0.16 p = 0.047). Although small sample size limits inference, these data support that PEG3 methylation status has potential as a molecular target for inclusion in CIN screening to improve prediction of progression. Impact statement: We present the first evidence that aberrant methylation of the PEG3 DMR is an important co-factor in the development of Invasive cervical carcinoma (ICC), especially among women infected with high risk HPV. Our results show that a five percent increase in DNA methylation of PEG3 is associated with a 1.6-fold increase ICC risk. Suggesting PEG3 methylation status may be useful as a molecular marker for CIN screening to improve prediction of cases likely to progress.

We developed a paclitaxel-conjugated polymeric micelle, ABP-PEG3.5k-Paclitaxel (APP) consisting of poly (ethylene glycol) (PEG) and arginine-grafted poly (cystaminebisacrylamide-diaminohexane) (ABP) for the co-delivery of gene and drug. The APP polymer self-assembled into cationic polymeric micelles with a critical micelle concentration (CMC) value of approximately 0.062 mg/mL, which was determined from measurements of the UV absorption of pyrene. The micelles have an average size of about 3 nm and a zeta potential of about +14 mV. Due to the positive surface charge, APP micelles formed polyplexes with plasmid DNA approximately 200 nm in diameter. The luciferase gene and mouse interleukin-12 (IL-12) gene was used to monitor gene delivery potency. APP polyplexes showed increased gene delivery efficiency and cellular uptake with higher anticancer potency than paclitaxel alone. These results demonstrate that an APP micelle-based delivery system is well suitable for the co-delivery of gene and drug.

Opisthorchis viverrini-related cholangiocarcinoma (CCA), a fatal bile duct cancer, is a major public health concern in areas endemic for this parasite. We report here whole-exome sequencing of eight O. viverrini-related tumors and matched normal tissue. We identified and validated 206 somatic mutations in 187 genes using Sanger sequencing and selected 15 genes for mutation prevalence screening in an additional 46 individuals with CCA (cases). In addition to the known cancer-related genes TP53 (mutated in 44.4% of cases), KRAS (16.7%) and SMAD4 (16.7%), we identified somatic mutations in 10 newly implicated genes in 14.8-3.7% of cases. These included inactivating mutations in MLL3 (in 14.8% of cases), ROBO2 (9.3%), RNF43 (9.3%) and PEG3 (5.6%), and activating mutations in the GNAS oncogene (9.3%). These genes have functions that can be broadly grouped into three biological classes: (i) deactivation of histone modifiers, (ii) activation of G protein signaling and (iii) loss of genome stability. This study provides insight into the mutational landscape contributing to O. viverrini-related CCA.

INTRODUCTION: In many normal tissues, proliferation rates decline postnatally, causing somatic growth to slow. Previous evidence suggests that this decline is due, in part, to decline in the expression of growth-promoting imprinted genes including Mest, Plagl1, Peg3, Dlk1, and Igf2. Embryonal cancers are composed of cells that maintain embryonic characteristics and proliferate rapidly in childhood. We hypothesized that the abnormal persistent rapid proliferation in embryonal cancers occurs in part because of abnormal persistent high expression of growth-promoting imprinted genes.
RESULTS: Analysis of microarray data showed elevated expression of MEST, PLAGL1, PEG3, DLK1, and IGF2 in various embryonal cancers, especially rhabdomyosarcoma, as compared to nonembryonal cancers and normal tissues. Similarly, mRNA expression, assessed by real-time PCR, of MEST, PEG3, and IGF2 in rhabdomyosarcoma cell lines was increased as compared to nonembryonal cancer cell lines. Furthermore, siRNA-mediated knockdown of MEST, PLAGL1, PEG3, and IGF2 expression inhibited proliferation in Rh30 rhabdomyosarcoma cells.
DISCUSSION: These findings suggest that the normal postnatal downregulation of growth-promoting imprinted genes fails to occur in some embryonal cancers, particularly rhabdomyosarcoma, and contributes to the persistent rapid proliferation of rhabdomyosarcoma cells and, more generally, that failure of the mechanisms responsible for normal somatic growth deceleration can promote tumorigenesis.

To identify epigenetic-based biomarkers for diagnosis of ovarian cancer we performed MeDIP-Chip in A2780 and CaOV3 ovarian cancer cell lines. Validation by Sequenom massARRAY methylation analysis confirmed a panel of six gene promoters (ARMCX1, ICAM4, LOC134466, PEG3, PYCARD & SGNE1) where hypermethylation discriminated 27 serous ovarian cancer clinical samples versus 12 normal ovarian surface epithelial cells (OSE) (ROC of 0.98). Notably, CpG sites across the transcription start site of a potential long-intergenic non-coding RNA (lincRNA) gene (LOC134466), was shown to be hypermethylated in 81% of serous EOC and could differentiate tumours from OSE (p<0.05). We propose that this potential biomarker panel holds great promise as a diagnostic test for high-grade (Type II) serous ovarian cancer.

BACKGROUND: Epigenetic therapy has had a significant impact on the management of hematologic malignancies, but its role in the treatment of ovarian cancer remains to be defined. The authors previously demonstrated that treatment of ovarian and breast cancer cells with DNA methyltransferase and histone deacetylase (HDAC) inhibitors can up-regulate the expression of imprinted tumor suppressors. In this study, demethylating agents and HDAC inhibitors were tested for their ability to induce re-expression of tumor suppressor genes, inhibiting growth of ovarian cancer cells in culture and in xenografts.
METHODS: Ovarian cancer cells (Hey and SKOv3) were treated with demethylating agents (5-aza-20-deoxycytidine [DAC] or 5-azacitidine [AZA]) or with HDAC inhibitors (suberoylanilide hydroxamicacid [SAHA] or trichostatin A [TSA]) to determine their impact on cellular proliferation, cell cycle regulation, apoptosis, autophagy, and re-expression of 2 growth inhibitory imprinted tumor suppressor genes: guanosine triphosphate-binding Di-RAS-like 3 (ARHI) and paternally expressed 3 (PEG3). The in vivo activities of DAC and SAHA were assessed in a Hey xenograft model.
RESULTS: The combination of DAC and SAHA produced synergistic inhibition of Hey and SKOv3 cell growth by apoptosis and cell cycle arrest. DAC induced autophagy in Hey cells that was enhanced by SAHA. Treatment with both agents induced re-expression of ARHI and PEG3 in cultured cells and in xenografts, correlating with growth inhibition. Knockdown of ARHI decreased DAC-induced autophagy. DAC and SAHA inhibited the growth of Hey xenografts and induced autophagy in vivo.
CONCLUSIONS: A combination of DAC and SAHA inhibited ovarian cancer growth while inducing apoptosis, G2/M arrest, autophagy, and re-expression of imprinted tumor suppressor genes.

Disruption of the normal pattern of parental origin-specific gene expression is referred to as loss of imprinting (LOI), which is common in various cancers. To investigate a possible role of LOI in the early stage of human cell transformation, we studied LOI in 18 human fibroblast cell lines immortalized spontaneously, by viral oncogenes, by chemical or physical carcinogens, or by infection with a retrovirus vector encoding the human telomerase catalytic subunit, hTERT cDNA. LOI was observed in all the 18 immortal cell lines. The gene most commonly exhibiting LOI was NDN which displayed LOI in 15 of the 18 cell lines (83%). The other genes exhibiting LOI at high frequencies were PEG3 (50%), MAGE-L2 (61%) and ZNF 127 (50%). Expression of NDN that was lost in the immortal cell lines was restored by treatment with 5-aza-2'-deoxycytidine. The ratio of histone H3 lysine 9 methylation to histone H3 lysine 4 methylation of the chromatin containing the NDN promoter in the immortal WI-38VA13 cells was greater than that in the parental cells, suggesting chromatin structure-mediated regulation of NDN expression. We previously demonstrated that inactivation of the p16INK4a/pRb pathway is necessary for immortalization of human cells. Human fibroblasts in the pre-crisis phase and cells with an extended lifespan that eventually senesce, both of which have the normal p16INK4a/pRb pathway, did not show LOI at any imprinted gene examined. Although it is not clear if LOI plays a causal role in immortalization of human cells or is merely coincidental, these findings indicate a possible involvement of LOI in immortalization of human cells or a common mechanism involved in both processes.

The imprinted gene PEG3 confers parenting and sexual behaviors, alters growth and development, and regulates apoptosis. However, a molecular mechanism that can account for the diverse functions of Peg3/Pw1 is not known. To elucidate Peg3-regulated pathways, we performed a functional screen in zebrafish. Enforced overexpression of PEG3 mRNA during zebrafish embryogenesis decreased beta-catenin protein expression and inhibited Wnt-dependent tail development. Peg3/Pw1 also inhibited Wnt signaling in human cells by binding to beta-catenin and promoting its degradation via a p53/Siah1-dependent, GSK3beta-independent proteasomal pathway. The inhibition of the Wnt pathway by Peg3/Pw1 suggested a role in tumor suppression. Hypermethylation of the PEG3 promoter in primary human gliomas led to a loss of imprinting and decreased PEG3 mRNA expression that correlated with tumor grade. The decrease in Peg3/Pw1 protein expression increased beta-catenin, promoted proliferation, and inhibited p53-dependent apoptosis in human CD133(+) glioma stem cells. Thus, mammalian imprinting utilizes Peg3/Pw1 to co-opt the Wnt pathway, thereby regulating development and glioma growth.

Glioma includes astrocytoma, oligodendroglioma, ependymoma and glioblastoma. We previously reported the epigenetic silencing of paternally expressed gene 3 (PEG3) in glioma cell lines. In this study, we investigated methylation of an exonic CpG island in the promoter region and the expression of PEG3 gene in 20 glioma and 5 non-tumor tissue samples. We found wide variations in the methylation level. Hypomethylaiton and hypermethylation was found in 3 and 4 glioma tissue samples, respectively. Monoallelic expression, which is an evidence of an imprinted gene, was maintained in eight out of nine informative cases which have T/C polymorphisms in PEG3. The lower gene expression, which suggested epigenetic silencing of PEG3, was confirmed statistically in glioblastoma using quantitative reverse-transcription polymerase chain reaction. Interestingly, we found higher expression of PEG3 in two out of three oligodendrogliomas. A negative correlation between the methylation level and gene expression was shown by regression analysis. These results suggest that the abnormal regulation of PEG3 is associated with several glioma subtypes and that it plays an important role in tumorigenesis.

Current treatment options for neuroblastoma fail to eradicate the disease in the majority of high-risk patients, clearly mandating development of innovative therapeutic strategies. Gene therapy represents a promising approach for reversing the neoplastic phenotype or driving tumor cells to self-destruction. We presently studied the effects of adenovirus-mediated gene transfer of human polynucleotide phosphorylase (hPNPase(old-35)), a 3',5'-exoribonuclease with growth-inhibitory properties, in neuroblastoma cells. Transgene expression was driven by either the cytomegalovirus (CMV) promoter or by a tumor-selective promoter derived from progression elevated gene-3 (PEG-3). Our data demonstrate that efficient adenoviral transduction of neuroblastoma cells and robust transgene expression are feasible objectives, that the PEG-3 promoter is capable of selectively targeting gene expression in the majority of neuroblastoma cells, and that hPNPase(old-35) induces profound growth suppression and apoptosis of malignant neuroblastoma cells, while exerting limited effects on normal neural crest-derived melanocytes. These findings support future applications of hPNPase(old-35) for targeted gene-based therapy of neuroblastoma and suggest that combination with the PEG-3 promoter holds promise for creating a potent and selective neuroblastoma therapeutic. J. Cell. Physiol. 219: 707-715, 2009. (c) 2009 Wiley-Liss, Inc.

It remains possible that chemicals that act by mutagenic mechanisms as well as chemicals that do not induce gene mutations may affect epigenetic gene expression. To test the possibility, we investigated the ability of both types of chemicals to alter the expression of five imprinted genes, PEG3, SNRPN, NDN, ZAC and H19, using two human colon cancer cell lines and a human breast cancer cell line. The expression of imprinted genes was changed by some non-mutagenic and mutagenic carcinogens independent of their mutagenic activity. The genes most commonly exhibiting the changes in expression were SNRPN and PEG3. Alterations of the expression of NDN and ZAC were also observed in some conditions. Methylation-specific PCR and chromatin immunoprecipitation assays suggest the possibility that changes in the expression of SNRPN may be associated with DNA hypomethylation and histone acetylation of the promoters and euchromatinization of the heterochromatic domains of the promoters. Changes in expression of the imprinted genes, PEG3 and NDN, were also observed in cells immortalized by treatment of normal human fibroblasts with 4-nitroquinoline 1-oxide or aflatoxin B1. We previously demonstrated that expression of the cancer-related gene, INK4a, in these immortal cells was lost via epigenetic mechanisms. The results prove that, in cancer cells, some mutagenic or non-mutagenic carcinogens can epigenetically influence the transcription levels of imprinted genes and also suggest the possibility that some chemical carcinogens may have epigenetic carcinogenic effects in human cells.

BACKGROUND: Imprinted tumor suppressor genes may be particularly important in the pathogenesis of ovarian cancer. Two imprinted genes, paternally expressed 3 (PEG3) and aplasia Ras homologue member I (ARHI), are the most frequently down-regulated in ovarian cancers on gene expression arrays.
METHODS: PEG3 and ARHI expression levels were evaluated with real-time reverse-transcriptase polymerase chain reaction (PCR) analysis. Promoter methylation was measured by pyrosequencing, and loss of heterozygosity (LOH) was detected by PCR-LOH assays.
RESULTS: PEG3 was down-regulated in 75% and ARHI was down-regulated in 88% of 40 ovarian cancers. ARHI CpG islands I and II were hypermethylated in 13 of 42 ovarian cancers (31%) and in 5 of 42 ovarian cancers (12%), respectively, and hypermethylation was associated with reduced ARHI expression in all 18 samples of ovarian cancer with CpG island hypermethylation. PEG3 was hypermethylated in 11 of 42 ovarian cancers (26%), and PEG3 expression was down-regulated in 10 of those 11 cancers. LOH was detected in 8 of 35 informative cases for ARHI (23%) and in 5 of 25 informative cases for PEG3 (20%). PEG3 and ARHI expression was highly correlated in human ovarian cancers (correlation coefficient [R]=0.69; P< .0001). PEG3 and ARHI also were methylated concordantly in ovarian cancers (R=0.36; P= .019). Re-expression of PEG3, similar to that of ARHI, markedly inhibited ovarian cancer growth. ARHI and PEG3 expression could be restored by treatment with 5-aza-2'-deoxycytidine and trichostatin A, consistent with the importance of promoter methylation and histone acetylation in regulating expression of both genes.
CONCLUSIONS: Loss of expression of the growth-inhibitory imprinted genes ARHI and PEG3 through promoter methylation, LOH, and other mechanisms may stimulate clonogenic growth and contribute to the pathogenesis of a majority of ovarian cancers.

A complete hydatidiform mole (CHM) is an abnormal pregnancy with hyperproliferative vesicular trophoblast and no fetal development. Most CHM are sporadic and androgenetic, but recurrent HM have biparental inheritance (BiHM) with disrupted DNA methylation at differentially methylated regions (DMRs) of imprinted loci. Some women with recurrent BiHM have mutations in the NLRP7 gene on chromosome 19q13.42. Using bisulfite genomic sequencing at eight imprinted DMRs on DNA from two BiHMs, we found a pattern of failure to acquire or maintain DNA methylation at DMRs (PEG3, SNRPN, KCNQ1OT1, GNAS exon 1A) that normally acquire CpG methylation during oogenesis, but not at H19, which acquires CpG methylation during spermatogenesis. Secondary imprints at the GNAS locus showed variable abnormal patterns with both gain and loss of CpG methylation. We found novel missense and splice-site mutations in NLRP7 in women with non-familial recurrent BiHM. We identified and characterized a homozygous intragenic tandem duplication including exons 2 through 5 of NLRP7 that results in a predicted truncated protein in affected women of three unrelated Egyptian kindreds, suggesting a founder effect. Our findings firmly establish that NLRP7 mutations are a major cause of BiHM and confirm presence of a complex pattern of imprinting abnormalities in BiHM tissues.

Disregulation of imprinted genes can be associated with tumorigenesis and altered cell differentiation capacity and so could provide adverse outcomes for stem cell applications. Although the maintenance of mouse and primate embryonic stem cells in a pluripotent state has been reported to disrupt the monoallelic expression of several imprinted genes, available data have suggested relatively higher imprint stability in the human equivalents. Identification of 202 heterozygous loci allowed us to examine the allelic expression of 22 imprinted genes in 22 human embryonic stem cell lines. Half of the genes examined (IPW, H19, MEG3, MEST isoforms 1 and 2, PEG10, MESTIT1, NESP55, ATP10A, PHLDA2, IGF2) showed variable allelic expression between lines, indicating vulnerability to disrupted imprinting. However, seven genes showed consistent monoallelic expression (NDN, MAGEL2, SNRPN, PEG3, KCNQ1, KCNQ1OT1, CDKN1C). Furthermore, four genes known to be monoallelic or to exhibit polymorphic imprinting in later-developing human tissues (TP73, IGF2R, WT1, SLC22A18) were always biallelic in hESCs. MEST isoform 1, PEG10, and NESP55 showed an association between the variability observed in interline allelic expression status and the DNA methylation of previously identified regulatory regions. Our results demonstrate gene-specific differences in the stability of imprinted loci in human embryonic stem cells and identify disrupted DNA methylation as one potential mechanism. We conclude the prudence of including comprehensive imprinting analysis in the continued characterization of human embryonic stem cell lines.

Muscle wasting (cachexia) is an incurable complication associated with chronic infection and cancers that leads to an overall poor prognosis for recovery. Tumor necrosis factor-alpha (TNFalpha) is a key inflammatory cytokine associated with cachexia. TNFalpha inhibits myogenic differentiation and skeletal muscle regeneration through downstream effectors of the p53 cell death pathway including PW1/Peg3, bax, and caspases. We report that p53 is required for the TNFalpha-mediated inhibition of myogenesis in vitro and contributes to muscle wasting in response to tumor load in vivo. We further demonstrate that PW1 and p53 participate in a positive feedback regulatory loop in vitro. Consistent with this observation, we find that the number of PW1-expressing stem cells in skeletal muscle declines significantly in p53 nullizygous mice. Furthermore, gene transfer of a dominant-negative form of PW1 into muscle tissue in vivo blocks myofiber atrophy in response to tumor load. Taken together, these results show a novel role for p53 in mediating muscle stem cell behavior and muscle atrophy, and point to new targets for the therapeutic treatment of muscle wasting.