Obtaining representative samples from a term placenta for gene-expression studies is confounded by both within placental heterogeneity and sampling effects such as sample location and processing time. Epigenetic processes involved in the regulation of gene expression, such as DNA methylation, may show similar variability, but are less well studied. Therefore, we investigated the nature of within and between- placenta variation in gene expression and DNA methylation of genes that were chosen for being differentially expressed or methylated by cell type within the placenta.

METHODS

In total, two or more samples from each of 38 normal term placentae were utilized. The expression levels of <em>CDH1</em>, <em>CDH1</em>1, ID2, PLAC1 and KISS1 were evaluated by real-time PCR. DNA methylation levels of LINE1 elements and CpGs within the promoter regions of KISS1, PTPN6, CASP8, and APC were similarly quantified by pyrosequencing.

RESULTS

Despite considerable sample-to-sample variability within each placenta, the within-placenta correlation for both gene expression and methylation was significant for each studied gene. Most of this variability was not due to sample location. However, between placental differences in gene expression were inflated by the dramatic effect of processing time (0-24 h) on mRNA levels, particularly for PLAC1 and KISS1 (both expressed in the apical syncytiotrophoblast). In contrast, DNA methylation levels remained relatively constant over this same time period.

CONCLUSIONS

Due to extensive site-to-site variability, multiple sampled sites are needed to accurately represent a placenta for molecular studies. Furthermore, mRNA quantitation of some genes may be hampered by its rapid degradation post-delivery (and possibly perinatally) and thus processing time should be considered in such analyses. Within-placenta correlations in expression and methylation from unrelated genes raise the possibility that methylation and expression variation may potentially reflect cell composition differences between samples rather than true differences occurring at the cellular level.

Meiotic maturation in oocytes is a prolonged process that is unique because of cell cycle arrests at prophase of meiosis I (MI) and at metaphase of meiosis II (MII). Fluctuations in cyclin-dependent kinase 1 (CDK1/CDC2A) activity govern meiotic progression, yet little is known about how these fluctuations are achieved. CDC14 is a highly conserved dual-specificity phosphatase that counteracts the function of proteins phosphorylated by CDK. Mammals contain two CDC14 homologs, CDC14A and CDC14B. We report that CDC14B localizes with the meiotic spindle in mouse oocytes, and (unlike somatic cells) it does not localize in the nucleolus. Oocytes that overexpress CDC14B are significantly delayed in resuming meiosis and fail to progress to MII, whereas oocytes depleted of CDC14B spontaneously resume meiosis under conditions that normally inhibit meiotic resumption. Depletion of FZR1 (CDH1), a regulatory subunit of the anaphase-promoting complex/cyclosome that targets cyclin B1 (CCNB1) for ubiquitin-mediated proteolysis, partially restores normal timing of meiotic resumption in oocytes with excess CDC14B. These studies also reveal that experimentally altering CDC14B levels generates eggs with abnormal spindles and with chromosome alignment perturbations. Our data indicate that CDC14B is a negative regulator of meiotic resumption and may regulate MI in mouse oocytes.

Human pituitary adenomas are the most common intracranial neoplasms. Approximately 5% of them are familial adenomas. Patients with familial tumors carry germline mutations in predisposition genes, including AIP, MEN1 and PRKAR1A. These mutations are extremely rare in sporadic pituitary adenomas, which therefore are caused by different mechanisms. Multiple tumor suppressive genes linked to sporadic tumors have been identified. Their inactivation is caused by epigenetic mechanisms, mainly promoter hypermethylation, and can be placed into two groups based on their functional interaction with tumor suppressors RB or p53. The RB group includes CDKN2A, CDKN2B, CDKN2C, RB1, BMP4, <em>CDH1</em>, <em>CDH1</em>3, GADD45B and GADD45G; AIP and MEN1 genes also belong to this group. The p53 group includes MEG3, MGMT, PLAGL1, RASSF1, RASSF3 and SOCS1. We propose that the tumor suppression function of these genes is mainly mediated by the RB and p53 pathways. We also discuss possible tumor suppression mechanisms for individual genes.

Hypermethylation of CpG islands is associated with silencing of various tumor suppressor genes. Recent studies on colorectal and gastric cancer have identified a CpG island methylator phenotype (CIMP), which involves the targeting of multiple genes by promoter hypermethylation. For determination of association between DNA methylation pattern or histological type and CIMP status in gastric carcinoma, CpG islands in the promoters of hMLH1 and CDH1 genes, CpG islands overlapping exon 1 of MGMT and p16(INK4a) genes, and a non-CpG island in exon 1 of the RAR-beta gene were studied. The presence of the CIMP was determined by monitoring five methylated in tumor (MINT ) loci in 103 gastric carcinomas. Among the 103 gastric carcinomas, DNA hypermethylation was detected in the following frequencies: 14 (14%) for hMLH1, 26 (25%) for MGMT, 26 (25%) for p16(INK4a), 54 (52%) for CDH1, and 53 (52%) for RAR-beta. Forty-two (41%) of 103 gastric carcinomas were positive for the CIMP. CIMP and hypermethylation of p16(INK4a) gene were found more frequently in intestinal and diffuse-adherent types than in diffuse-scattered type (P = 0.013 and 0.017, respectively). In contrast, hypermethylation of the CDH1 and RAR-beta genes was more common in the diffuse-scattered type than in the other types (P = 0.008 and 0.007, respectively). In intestinal- and diffuse-adherent-type gastric carcinomas, we found significant associations between the presence of the CIMP and hypermethylation of several genes: hMLH1 (P = 0.006), p16(INK4a) (P = 0.018), CDH1 (P = 0.024), and RAR-beta (P = 0.044). Our overall results suggest that in some intestinal- and diffuse-adherent-type gastric carcinomas, DNA hypermethylation affects non-specific gene promoters concordantly, at least in part, whereas in diffuse-scattered-type gastric carcinoma, DNA hypermethylation affects specific genes such as CDH1 and RAR-beta.

BACKGROUND

Gastric cancer is the second-leading cause of global cancer deaths, with metastatic disease representing the primary cause of mortality. To identify candidate drivers involved in oncogenesis and tumor evolution, we conduct an extensive genome sequencing analysis of metastatic progression in a diffuse gastric cancer. This involves a comparison between a primary tumor from a hereditary diffuse gastric cancer syndrome proband and its recurrence as an ovarian metastasis.

RESULTS

Both the primary tumor and ovarian metastasis have common biallelic loss-of-function of both the CDH1 and TP53 tumor suppressors, indicating a common genetic origin. While the primary tumor exhibits amplification of the Fibroblast growth factor receptor 2 (FGFR2) gene, the metastasis notably lacks FGFR2 amplification but rather possesses unique biallelic alterations of Transforming growth factor-beta receptor 2 (TGFBR2), indicating the divergent in vivo evolution of a TGFBR2-mutant metastatic clonal population in this patient. As TGFBR2 mutations have not previously been functionally validated in gastric cancer, we modeled the metastatic potential of TGFBR2 loss in a murine three-dimensional primary gastric organoid culture. The Tgfbr2 shRNA knockdown within Cdh1-/-; Tp53-/- organoids generates invasion in vitro and robust metastatic tumorigenicity in vivo, confirming Tgfbr2 metastasis suppressor activity.

CONCLUSIONS

We document the metastatic differentiation and genetic heterogeneity of diffuse gastric cancer and reveal the potential metastatic role of TGFBR2 loss-of-function. In support of this study, we apply a murine primary organoid culture method capable of recapitulating in vivo metastatic gastric cancer. Overall, we describe an integrated approach to identify and functionally validate putative cancer drivers involved in metastasis.

Diffuse gastric cancers typically present as late-stage tumours and, as a result, the 5 year survival rate is poor. Some gastric cancers are hereditary and these tend to be of the diffuse type; 30-40% of hereditary diffuse gastric cancers (HDGCs) can be explained by defective germline alleles of E-cadherin (CDH1), but for the remaining families the factors driving susceptibility remain unknown. We had access to a large HDGC pedigree with no obvious mutation in CDH1, and applied exome sequencing to identify new genes involved in gastric cancer. We identified a germline truncating allele of α-E-catenin (CTNNA1) that was present in two family members with invasive diffuse gastric cancer and four in which intramucosal signet ring cells were detected as part of endoscopic surveillance. The remaining CTNNA1 allele was silenced in the two diffuse gastric cancers from the family that were available for screening, and this was also true for signet ring cells identified in endoscopic biopsies. Since α-E-catenin functions in the same complex as E-cadherin, our results call attention to the broader signalling network surrounding these proteins in HDGC. We also detected somatic mutations in one tumour and found substantial overlap with genes mutated in sporadic gastric cancer, including PIK3CA, ARID1A, MED12 and MED23.

Recent sequencing analyses have shed light on heterogeneous patterns of genomic aberrations in human gastric cancers (GCs). To explore how individual genetic events translate into cancer phenotypes, we established a biological library consisting of genetically engineered gastric organoids carrying various GC mutations and 37 patient-derived organoid lines, including rare genomically stable GCs. Phenotype analyses of GC organoids revealed divergent genetic and epigenetic routes to gain Wnt and R-spondin niche independency. An unbiased phenotype-based genetic screening identified a significant association between CDH1/TP53 compound mutations and the R-spondin independency that was functionally validated by CRISPR-based knockout. Xenografting of GC organoids further established the feasibility of Wnt-targeting therapy for Wnt-dependent GCs. Our results collectively demonstrate that multifaceted genetic abnormalities render human GCs independent of the stem cell niche and highlight the validity of the genotype-phenotype screening strategy in gaining deeper understanding of human cancers.

BACKGROUND

Epithelial-mesenchymal transition (EMT) is known to impart metastasis and stemness characteristics in breast cancer. To characterize the epigenetic reprogramming following Twist1-induced EMT, we characterized the epigenetic and transcriptome landscapes using whole-genome transcriptome analysis by RNA-seq, DNA methylation by digital restriction enzyme analysis of methylation (DREAM) and histone modifications by CHIP-seq of H3K4me3 and H3K27me3 in immortalized human mammary epithelial cells relative to cells induced to undergo EMT by Twist1.

RESULTS

EMT is accompanied by focal hypermethylation and widespread global DNA hypomethylation, predominantly within transcriptionally repressed gene bodies. At the chromatin level, the number of gene promoters marked by H3K4me3 increases by more than one fifth; H3K27me3 undergoes dynamic genomic redistribution characterized by loss at half of gene promoters and overall reduction of peak size by almost half. This is paralleled by increased phosphorylation of EZH2 at serine 21. Among genes with highly altered mRNA expression, 23.1% switch between H3K4me3 and H3K27me3 marks, and those point to the master EMT targets and regulators CDH1, PDGFRα and ESRP1. Strikingly, Twist1 increases the number of bivalent genes by more than two fold. Inhibition of the H3K27 methyltransferases EZH2 and EZH1, which form part of the Polycomb repressive complex 2 (PRC2), blocks EMT and stemness properties.

CONCLUSIONS

Our findings demonstrate that the EMT program requires epigenetic remodeling by the Polycomb and Trithorax complexes leading to increased cellular plasticity. This suggests that inhibiting epigenetic remodeling and thus decrease plasticity will prevent EMT, and the associated breast cancer metastasis.

BACKGROUND

The frequency of E-cadherin germline mutations in countries with different incidence rates for gastric carcinoma has not been well established. The goal of this study was to assess the worldwide frequency of CDH1 germline mutations in gastric cancers coming from low- and high-risk areas.

METHODS

English articles using MEDLINE access (from 1998 to 2011). Search terms included CDH1, E-cadherin, germline mutation, gastric cancer, hereditary, familial and diffuse histotype.The study included all E-cadherin germline mutations identified in gastric cancer patients; somatic mutations and germline mutations reported in other tumors were excluded.The method of this study was scheduled in accordance with the "PRISMA statement for reporting systematic reviews and meta-analyses". Countries were classified as low- or middle/high risk-areas for gastric carcinoma incidence. Statistical analysis was performed to correlate the CDH1 mutation frequency with gastric cancer incidence areas.

RESULTS

A total of 122 E-cadherin germline mutations have been identified; the majority (87.5%) occurred in gastric cancers coming from low-risk areas. In high-risk areas, we identified 16 mutations in which missense mutations were predominant. (68.8%). We verified a significant association between the mutation frequency and the gastric cancer risk area (p < 0.001: overall identified mutations in low- vs. middle/high-risk areas).

CONCLUSIONS

E-cadherin genetic screenings performed in low-risk areas for gastric cancer identified a higher frequency of CDH1 germline mutations. This data could open new approaches in the gastric cancer prevention test; before proposing a proband candidate for the CDH1 genetic screening, geographic variability, alongside the family history should be considered.

Approximately one-third of prostate cancer patients present with intermediate risk disease. Interestingly, while this risk group is clinically well defined, it demonstrates the most significant heterogeneity in PSA-based biochemical outcome. Further, the majority of candidate genes associated with prostate cancer progression have been identified using cell lines, xenograft models, and high-risk androgen-independent or metastatic patient samples. We used a global high-resolution array comparative genomic hybridization (CGH) assay to characterize copy number alterations (CNAs) in intermediate risk prostate cancer. Herein, we show this risk group contains a number of alterations previously associated with high-risk disease: (1) deletions at 21q22.2 (TMPRSS2:ERG), 16q22-24 (containing CDH1), 13q14.2 (RB1), 10q23.31 (PTEN), 8p21 (NKX3.1); and, (2) amplification at 8q21.3-24.3 (containing c-MYC). In addition, we identified six novel microdeletions at high frequency: 1q42.12-q42.3 (33.3%), 5q12.3-13.3 (21%), 20q13.32-13.33 (29.2%), 22q11.21 (25%), 22q12.1 (29.2%), and 22q13.31 (33.3%). Further, we show there is little concordance between CNAs from these clinical samples and those found in commonly used prostate cancer cell models. These unexpected findings suggest that the intermediate-risk category is a crucial cohort warranting further study to determine if a unique molecular fingerprint can predict aggressive versus indolent phenotypes.

The histone H3K27 demethylase, UTX, is a known component of the H3K4 methyltransferase MLL complex, but its functional association with H3K4 methylation in human cancers remains largely unknown. Here we demonstrate that UTX loss induces epithelial-mesenchymal transition (EMT)-mediated breast cancer stem cell (CSC) properties by increasing the expression of the SNAIL, ZEB1 and ZEB2 EMT transcription factors (EMT-TFs) and of the transcriptional repressor CDH1. UTX facilitates the epigenetic silencing of EMT-TFs by inducing competition between MLL4 and the H3K4 demethylase LSD1. EMT-TF promoters are occupied by c-Myc and MLL4, and UTX recognizes these proteins, interrupting their transcriptional activation function. UTX decreases H3K4me2 and H3 acetylation at these promoters by forming a transcriptional repressive complex with LSD1, HDAC1 and DNMT1. Taken together, our findings indicate that UTX is a prominent tumour suppressor that functions as a negative regulator of EMT-induced CSC-like properties by epigenetically repressing EMT-TFs.

OBJECTIVE

Somatotropinomae are classified as densely and sparsely granulated adenomae, which typically exhibit a perinuclear pattern (PP) and a dot pattern (DP) in cytokeratin (CK) immunostaining respectively. Some exhibit a mixed pattern (MP). We studied the relationship between these somatotropinoma subtypes and their clinico-pathological features.

METHODS

The study population consisted of 141 Japanese acromegalic patients. We evaluated their clinical presentation and their response to provocation tests with TRH and LHRH and to suppression (octreotide) test. Tumour tissues were subjected to immunostaining for CAM-5.2, MIB-1, CD34, E-cadherin (CDH1) and p53 (TP53). In 43 cases (30 non-DP and 13 DP), we analysed gsp mutations (constitutively activating mutations of the G(s)α protein that is encoded by GNAS gene).

RESULTS

The 141 adenomae were categorised into three subtypes based on their CK staining patterns; 30 (21.3%) exhibited DP, 83 (58.9%) exhibited PP, and 28 (19.9%) exhibited MP. Compared with the other subtypes, DP adenomae were significantly larger, and their E-cadherin expression and response to TRH, LHRH and octreotide challenge were lower. The postoperative cure rate tended to be lower in DP adenomae. gsp mutations were detected in 25 of 43 cases examined (58.1%); 20 of the 30 non-DP (66.7%) and 5 of the 13 DP tumours (38.5%) were affected by the mutation.

CONCLUSIONS

DP somatotropinomae exhibit characteristic features. Compared with the non-DP subtypes, DP adenomae manifested a larger tumour size, a lower incidence of abnormal responses to TRH and LHRH challenge, a poor response to octreotide test and a lower expression of E-cadherin. gsp mutation was not exclusive for non-DP somatotropinomae.

Gastric cancer is not a single disease, and its subtype classification is still evolving. Next-generation sequencing studies have identified novel genetic drivers of gastric cancer, but their use as molecular classifiers or prognostic markers of disease outcome has yet to be established. In this study, we integrated somatic mutational profiles and clinicopathologic information from 544 gastric cancer patients from previous genomic studies to identify significantly mutated genes (SMG) with prognostic relevance. Gastric cancer patients were classified into regular (86.8%) and hypermutated (13.2%) subtypes based on mutation burden. Notably, TpCpW mutations occurred significantly more frequently in regular, but not hypermutated, gastric cancers, where they were associated with APOBEC expression. In the former group, six previously unreported (XIRP2, NBEA, COL14A1, CNBD1, ITGAV, and AKAP6) and 12 recurrent mutated genes exhibited high mutation prevalence (≥3.0%) and an unexpectedly higher incidence of nonsynonymous mutations. We also identified two molecular subtypes of regular-mutated gastric cancer that were associated with distinct prognostic outcomes, independently of disease staging, as confirmed in a distinct patient cohort by targeted capture sequencing. Finally, in diffuse-type gastric cancer, CDH1 mutation was found to be associated with shortened patient survival, independently of disease staging. Overall, our work identified previously unreported SMGs and a mutation signature predictive of patient survival in newly classified subtypes of gastric cancer, offering opportunities to stratify patients into optimal treatment plans based on molecular subtyping. Cancer Res; 76(7); 1724-32. ©2016 AACR.

Exposure to tobacco smoke is associated with increased DNA methylation at certain genes in both lung and bladder tumors. We sought to identify interactions in bladder cancer between DNA methylation and a history of smoking, along with any possible effect of aging. We measured DNA methylation in 342 transitional cell carcinoma tumors at BCL2, PTGS2 (COX2), DAPK, CDH1 (ECAD), EDNRB, RASSF1A, RUNX3, TERT, and TIMP3. The prevalence of methylation at RUNX3, a polycomb target gene, increased as a function of age at diagnosis (P = 0.031) and a history of smoking (P = 0.015). RUNX3 methylation also preceded methylation at the other eight genes (P < 0.001). It has been proposed that DNA methylation patterns constitute a "molecular clock" and can be used to determine the "age" of normal tissues (i.e., the number of times the cells have divided). Because RUNX3 methylation increases with age, is not present in normal urothelium, and occurs early in tumorigenesis, it can be used for the first time as a molecular clock to determine the age of a bladder tumor. Doing so reveals that tumors from smokers are "older" than tumors from nonsmokers (P = 0.009) due to tumors in smokers either initiating earlier or undergoing more rapid cell divisions. Because RUNX3 methylation is acquired early on in tumorigenesis, then its detection in biopsy or urine specimens could provide a marker to screen cigarette smokers long before any symptoms of bladder cancer are present.

Meiotic cohesin serves in sister chromatid linkage and DNA repair until its subunit Rec8 is cleaved by separase. Separase is activated when its inhibitor, securin, is polyubiquitinated by the Cdc20 regulated anaphase-promoting complex (APC(Cdc20)) and consequently degraded. Differently regulated APCs (APC(Cdh1), APC(Ama1)) have not been implicated in securin degradation at meiosis I. We show that Mnd2, a factor known to associate with APC components, prevents premature securin degradation in meiosis by APC(Ama1). mnd2Delta cells lack linear chromosome axes and exhibit precocious sister chromatid separation, but deletion of AMA1 suppresses these defects. Besides securin, Sgo1, a protein essential for protection of centromeric cohesion during anaphase I, is also destabilized in mnd2delta cells. Mnd2's disappearance prior to anaphase II may activate APC(Ama1). Human oocytes may spend many years in meiotic prophase before maturation. Inhibitors of meiotic APC variants could prevent loss of chiasmata also in these cells, thereby guarding against aberrant chromosome segregation.

Homeostatic maintenance of cellular mitochondria requires a dynamic balance between fission and fusion, and controlled changes in morphology are important for processes such as apoptosis and cellular division. Interphase mitochondria have been described as an interconnected network that fragments as cells enter mitosis, and this mitotic mitochondrial fragmentation is known to be regulated by the dynamin-related GTPase Drp1 (dynamin-related protein 1), a key component of the mitochondrial division machinery. Loss of Drp1 function and the subsequent failure of mitochondrial division during mitosis lead to incomplete cytokinesis and the unequal distribution of mitochondria into daughter cells. During mitotic exit and interphase, the mitochondrial network reforms. Here we demonstrate that changes in mitochondrial dynamics as cells exit mitosis are driven in part through ubiquitylation of Drp1, catalyzed by the APC/C(Cdh1) (anaphase-promoting complex/cyclosome and its coactivator Cdh1) E3 ubiquitin ligase complex. Importantly, inhibition of Cdh1-mediated Drp1 ubiquitylation and proteasomal degradation during interphase prevents the normal G1 phase regrowth of mitochondrial networks following cell division.

BACKGROUND

Aberrant promoter hypermethylation of cancer-associated genes occurs frequently during carcinogenesis and may serve as a cancer biomarker. In this study we aimed at defining a quantitative gene promoter methylation panel that might identify the most prevalent types of renal cell tumors.

METHODS

A panel of 18 gene promoters was assessed by quantitative methylation-specific PCR (QMSP) in 85 primarily resected renal tumors representing the four major histologic subtypes (52 clear cell (ccRCC), 13 papillary (pRCC), 10 chromophobe (chRCC), and 10 oncocytomas) and 62 paired normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters.

RESULTS

Significant differences in methylation levels among the four subtypes of renal tumors were found for CDH1 (p = 0.0007), PTGS2 (p = 0.002), and RASSF1A (p = 0.0001). CDH1 hypermethylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (p = 0.00016 and p = 0.0034, respectively), whereas PTGS2 methylation levels were significantly higher in ccRCC compared to pRCC (p = 0.004). RASSF1A methylation levels were significantly higher in pRCC than in normal tissue (p = 0.035). In pRCC, CDH1 and RASSF1A methylation levels were inversely correlated with tumor stage (p = 0.031) and nuclear grade (p = 0.022), respectively.

CONCLUSIONS

The major subtypes of renal epithelial neoplasms display differential aberrant CDH1, PTGS2, and RASSF1A promoter methylation levels. This gene panel might contribute to a more accurate discrimination among common renal tumors, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses.

Cdc7 kinase regulates DNA replication. However, its role in DNA repair and recombination is poorly understood. Here we describe a pathway that stabilizes the human Cdc7-ASK (activator of S-phase kinase; also called Dbf4), its regulation, and its function in cellular responses to compromised DNA replication. Stalled DNA replication evoked stabilization of the Cdc7-ASK (Dbf4) complex in a manner dependent on ATR-Chk1-mediated checkpoint signaling and its interplay with the anaphase-promoting complex/cyclosome(Cdh1) (APC/C(Cdh1)) ubiquitin ligase. Mechanistically, Chk1 kinase inactivates APC/C(Cdh1) through degradation of Cdh1 upon replication block, thereby stabilizing APC/C(Cdh1) substrates, including Cdc7-ASK (Dbf4). Furthermore, motif C of ASK (Dbf4) interacts with the N-terminal region of RAD18 ubiquitin ligase, and this interaction is required for chromatin binding of RAD18. Impaired interaction of ASK (Dbf4) with RAD18 disables foci formation by RAD18 and hinders chromatin loading of translesion DNA polymerase η. These findings define a novel mechanism that orchestrates replication checkpoint signaling and ubiquitin-proteasome machinery with the DNA damage bypass pathway to guard against replication collapse under conditions of replication stress.

CDH1 is a cell-cell adhesion molecule expressed in the epithelium to coordinate key morphogenetic processes, establish cell polarity, and regulate epithelial differentiation and proliferation. To determine the role of CDH1 in the mouse uterus, Cdh1 was conditionally ablated by crossing Pgr-Cre and Cdh1-flox mice, and the phenotype was characterized. We found that loss of Cdh1 results in a disorganized cellular structure of the epithelium and ablation of endometrial glands in the neonatal uterus. Cdh1(d/d) mice lost adherens junctions (CTNNB1 and CTNNA1) and tight junctions (claudin, occludin, and ZO-1 proteins) in the neonatal uterus, leading to loss of epithelial cell-cell interaction. Ablation of Cdh1 induced abnormal epithelial proliferation and massive apoptosis, and disrupted Wnt and Hox gene expression in the neonatal uterus. Although the uteri of Cdh1(d/d) mice did not show any myometrial defects, ablation of Cdh1 inhibited expression of epithelial (cytokeratin 8) and stromal (CD10) markers. Cdh1(d/d) mice were infertile because of defects during implantation and decidualization. Furthermore, we showed in the model of conditional ablation of both Cdh1 and Trp53 in the uterus that interrupting cell cycle regulation through the loss of Cdh1 leads to abnormal uterine development. The uteri of Cdh1(d/d) Trp53(d/d) mice exhibited histological features of endometrial carcinomas with myometrial invasion. Collectively, these findings suggest that CDH1 has an important role in structural and functional development of the uterus as well as adult uterine function. CDH1 has a capacity to control cell fate by altering directional cell proliferation and apoptosis.

Genetic factors, Helicobacter pylori infection, salt over-uptake, decreased vegetable/fruit consumption, smoking, and metabolic syndrome are risk factors of human gastric cancer. Germline mutations of CDH1 gene, and SNPs of PTPN11 (SHP2), TLR4, IL1B, TNFA, BMP6, GDF15 and RUNX3 genes are associated with gastric cancer. Helicobacter pylori activates CagA-SHP2-ERK and peptidoglycan-NOD1-NFkappaB signaling cascades in gastric epithelial cells using type IV secretion system, and also TRAF6-MAP3K7-NFkappaB and TRAF6-MAP3K7-AP-1 signaling cascades in epithelial and immune cells through lipopolysaccharide recognition by TLR2 or TLR4. IL-1beta, IL-6, IL-8, TNFalpha and IFNgamma are elevated in gastric mucosa with Helicobacter pylori infection. IL-6 and TNFalpha induce upregulation of WNT5A and WNT10B, respectively. WNT signals are transduced to beta-catenin-TCF/LEF, RhoA, JNK, PKC, NFAT, and NLK signaling cascades. WNT-beta-catenin-TCF/LEF signaling induces upregulation of MYC, CCND1, WISP1, FGF20, JAG1 and DKK1 genes. Notch signals are transduced to CSL-NICD-MAML and NFkappaB signaling cascades. FGF signals are transduced to ERK, PI3K-AKT, PKC, and NFAT signaling cascades. Helicobacter pylori infection induces SHH upregulation in parietal cell lineage, while BMP signals induce IHH upregulation in pit cell lineage. Hedgehog signals induce upregulation of GLI1, PTCH1, CCND2, FOXL1, JAG2 and SFRP1 genes. JAG1 and JAG2 activate Notch signaling, while DKK1 and SFRP1 inhibit WNT signaling. Stem cell signaling network, consisting of WNT, Notch, FGF, Hedgehog and BMP signaling pathways, is activated during chronic Helicobacter pylori infection. Epigenetic silencing of SFRP1 gene occurs in the earlier stage of carcinogenesis in the stomach, while amplification and overexpression of FGFR2 gene in the later stage. Dysregulation of the stem cell signaling network due to the accumulation of germline mutation, SNP, Helicobacter pylori infection, epigenetic change and genetic alteration gives rise to gastric cancer. SNP typing and custom-made microarray analyses on genes encoding stem cell signaling molecules could be utilized for the personalized medicine.

While most invasive breast cancers consist of carcinomas of the ductal type, about 10% are invasive lobular carcinomas. Invasive lobular and ductal carcinomas differ with respect to risk factors. Invasive lobular carcinoma is more strongly associated with exposure to female hormones, and therefore its incidence is more subject to variation. This is illustrated by US figures during the 1987 to 2004 period: after 12 years of increases, breast cancer incidence declined steadily from 1999 to 2004, reflecting among other causes the decreasing use of menopausal hormone therapy, and these variations were stronger for invasive lobular than for invasive ductal carcinoma. Similarly, invasive lobular carcinoma is more strongly associated with early menarche, late menopause and late age at first birth. As for genetic risk factors, four high-penetrance genes are tested in clinical practice when genetic susceptibility to breast cancer is suspected, BRCA1, BRCA2, TP53 and CDH1. Germline mutations in BRCA1 and TP53 are predominantly associated with invasive ductal carcinoma, while BRCA2 mutations are associated with both ductal and lobular cancers. CDH1, the gene coding for the E-cadherin adhesion protein, is of special interest as mutations are associated with invasive lobular carcinoma, but never with ductal carcinoma. It was initially known as the main susceptibility gene for gastric cancer of the diffuse type, but the excess of breast cancers of the lobular type in CDH1 families led researchers to identify it also as a susceptibility gene for invasive lobular carcinoma. The risk of invasive lobular carcinoma is high in female mutation carriers, as about 50% are expected to develop the disease. Carriers must therefore undergo intensive breast cancer screening, with, for example, yearly magnetic resonance imaging and mammogram starting at age 30 years.

OBJECTIVE

This study aims to quantitatively assess promoter and global methylation changes in pheochromocytomas and abdominal paragangliomas and its relation to tumor phenotypes.

METHODS

A panel of 53 primary tumors (42 benign, 11 malignant) was analyzed by quantitative bisulfite pyrosequencing. Based on methylation levels in the tumor suppressor genes, p16(INK4A), CDH1, DCR2, RARB, RASSF1A, NORE1A, TP73, APC, DAPK1, p14(ARF), and PTEN, a CpG island methylator phenotype (CIMP) was defined as concerted hypermethylation in three or more genes. Mean Z scores for the hypermethylated promoters were calculated to characterize overall promoter methylation. Global DNA methylation was quantified for LINE-1 promoter sequences and by using luminescent methylation analysis.

RESULTS

Five primary tumors (9.4%) exhibited a CIMP phenotype, four of which were malignant paragangliomas. CIMP was significantly associated with malignant behavior (P = 0.005) and younger age at presentation (P < 0.007) but did not result from BRAF V600E mutation. Global hypomethylation of LINE-1 elements was observed in tumors compared with normal adrenal samples (P < 0.02).

CONCLUSIONS

We here describe the identification of CIMP in abdominal paragangliomas and a strong association of this phenotype with malignant behavior, as well as young age at presentation. The findings raise a prospective for potential benefits of epigenetically acting drugs for a subgroup of young abdominal paraganglioma patients with adverse prognosis.

Abnormal expression of cadherins and catenins plays a critical role in the initiation and progression of multiple human tumours. This study aimed to evaluate the immunoreactivity of E- and P-cadherin, beta- and gamma-catenin, and p120ctn in premalignant and malignant endometrial lesions and to correlate their membranous expression with clinicopathological features. In addition, we examined whether or not LOH and promoter hypermethylation of the CDH1 gene were associated with E-cadherin expression and clinicopathological variables. Finally, we studied the frequency of beta-catenin mutations in premalignant endometrial lesions. Immunohistochemical staining was performed in 21 atypical endometrial hyperplasias (AEHs), 95 endometrioid carcinomas (EECs), and 33 non-endometrioid carcinomas (NEECs). Reduced E-cadherin expression was observed in 57.8% of the cases, being more frequent in NEECs (87.1%, p = 0.001) and carcinomas of more advanced stage (85.7% of stage III-IV carcinomas, p = 0.01). LOH of CDH1 gene was found in 57.1% of NEECs but only in 22.5% of EECs (p = 0.011) and showed a trend towards association with reduced E-cadherin expression (p = 0.089). CDH1 promoter hypermethylation was found in 21.2% of endometrial carcinomas but was not associated with clinicopathological or immunohistochemical variables. Reduced expression of beta- and gamma-catenin and p120ctn was found in 76.1%, 94.3%, and 63.6% of the cases, respectively, being more frequent in lesions with reduced E-cadherin expression. In addition, beta-catenin, but not gamma-catenin or p120ctn expression, was associated with the histology of the lesion, since it was reduced in 35% of AEHs, 80.3% of EECs, and 96.9% of NEECs (p = 0.000). Mutations in exon 3 of the beta-catenin gene, associated with beta-catenin nuclear expression, were detected in 3 (14.0%) AEH, a frequency similar to that previously reported in this series of ECs. Finally, upregulation of P-cadherin was observed in 28.6% of cases. This alteration was associated with the histology of the lesion, since it was found in 9.5% of AEHs, 27.7% of EECs, and 46.2% of NEECs (p = 0.021).

BACKGROUND

Stomach cancer is the third deadliest among all cancers worldwide. Although incidence of the intestinal-type gastric cancer has decreased, the incidence of diffuse-type is still increasing and its progression is notoriously aggressive. There is insufficient information on genome variations of diffuse-type gastric cancer because its cells are usually mixed with normal cells, and this low cellularity has made it difficult to analyze the genome.

RESULTS

We analyze whole genomes and corresponding exomes of diffuse-type gastric cancer, using matched tumor and normal samples from 14 diffuse-type and five intestinal-type gastric cancer patients. Somatic variations found in the diffuse-type gastric cancer are compared to those of the intestinal-type and to previously reported variants. We determine the average exonic somatic mutation rate of the two types. We find associated candidate driver genes, and identify seven novel somatic mutations in CDH1, which is a well-known gastric cancer-associated gene. Three-dimensional structure analysis of the mutated E-cadherin protein suggests that these new somatic mutations could cause significant functional perturbations of critical calcium-binding sites in the EC1-2 junction. Chromosomal instability analysis shows that the MDM2 gene is amplified. After thorough structural analysis, a novel fusion gene TSC2-RNF216 is identified, which may simultaneously disrupt tumor-suppressive pathways and activate tumorigenesis.

CONCLUSIONS

We report the genomic profile of diffuse-type gastric cancers including new somatic variations, a novel fusion gene, and amplification and deletion of certain chromosomal regions that contain oncogenes and tumor suppressors.