The DNA methylomes of serous borderline tumors reveal subgroups with malignant- or benign-like profiles.

Serous borderline tumors (SBOTs) are a challenging group of ovarian tumors positioned between benign and malignant disease. We have profiled the DNA methylomes of 12 low-grade serous carcinomas (LGSCs), 19 SBOTs, and 16 benign serous tumors (BSTs) across 27,578 CpG sites to further characterize the epigenomic relationship between these subtypes of ovarian tumors. Unsupervised hierarchical clustering of DNA methylation levels showed that LGSCs differ distinctly from BSTs, but not from SBOTs. Gene ontology analysis of genes showing differential methylation at linked CpG sites between LGSCs and BSTs revealed significant enrichment of gene groups associated with cell adhesion, cell-cell signaling, and the extracellular region, consistent with a more invasive phenotype of LGSCs compared with BSTs. Consensus clustering highlighted differences between SBOT methylomes and returned subgroups with malignant- or benign-like methylation profiles. Furthermore, a two-loci DNA methylation signature can distinguish between these SBOT subgroups with benign- and malignant-like methylation characteristics. Our findings indicate striking similarities between SBOT and LGSC methylomes, supporting a common origin and the view that LGSC may arise from SBOT. A subgroup of SBOTs can be classified into tumors with a benign- or a malignant-like methylation profile that may help in identifying tumors more likely to progress into LGSCs.

Frequency of mutations and polymorphisms in borderline ovarian tumors of known cancer genes.

Borderline ovarian tumors represent an understudied subset of ovarian tumors. Most studies investigating aberrations in borderline tumors have focused on KRAS/BRAF mutations. In this study, we conducted an extensive analysis of mutations and single-nucleotide polymorphisms (SNPs) in borderline ovarian tumors. Using the Sequenom MassArray platform, we investigated 160 mutations/polymorphisms in 33 genes involved in cell signaling, apoptosis, angiogenesis, cell cycle regulation and cellular senescence. Of 52 tumors analyzed, 33 were serous, 18 mucinous and 1 endometrioid. KRAS c.35G>A p.Gly12Asp mutations were detected in eight tumors (six serous and two mucinous), BRAF V600E mutations in two serous tumors, and PIK3CA H1047Y and PIK3CA E542K mutations in a serous and an endometrioid BOT, respectively. CTNNB1 mutation was detected in a serous tumor. Potentially functional polymorphisms were found in vascular endothelial growth factor (VEGF), ABCB1, FGFR2 and PHLPP2. VEGF polymorphisms were the most common and detected at four loci. PHLPP2 polymorphisms were more frequent in mucinous as compared with serous tumors (P=0.04), with allelic imbalance in one case. This study represents the largest and most comprehensive analysis of mutations and functional SNPs in borderline ovarian tumors to date. At least 25% of borderline ovarian tumors harbor somatic mutations associated with potential response to targeted therapeutics.

The role of arachidonic acid and its metabolites in insulin secretion from human islets of langerhans.

The roles played by arachidonic acid and its cyclooxygenase (COX)-generated and lipoxygenase (LOX)-generated metabolites have been studied using rodent islets and insulin-secreting cell lines, but very little is known about COX and LOX isoform expression and the effects of modulation of arachidonic acid generation and metabolism in human islets. We have used RT-PCR to identify mRNAs for cytosolic phospholipase A(2) (cPLA(2)), COX-1, COX-2, 5-LOX, and 12-LOX in isolated human islets. COX-3 and 15-LOX were not expressed by human islets. Perifusion experiments with human islets indicated that PLA(2) inhibition inhibited glucose-stimulated insulin secretion, whereas inhibitors of COX-2 and 12-LOX enzymes enhanced basal insulin secretion and also secretory responses induced by 20 mmol/l glucose or by 50 mumol/l arachidonic acid. Inhibition of COX-1 with 100 mumol/l acetaminophen did not significantly affect glucose-stimulated insulin secretion. These data indicate that the stimulation of insulin secretion from human islets in response to arachidonic acid does not require its metabolism through COX-2 and 5-/12-LOX pathways. The products of COX-2 and LOX activities have been implicated in cytokine-mediated damage of beta-cells, so selective inhibitors of these enzymes would be expected to have a dual protective role in diabetes: they would minimize beta-cell dysfunction while maintaining insulin secretion through enhancing endogenous arachidonic acid levels.

A role for the extracellular calcium-sensing receptor in cell-cell communication in pancreatic islets of langerhans.

BACKGROUND: The extracellular calcium-sensing receptor (CaR) is expressed in many tissues that are not associated with Ca(2+) homeostasis, including the endocrine cells in pancreatic islets of Langerhans. We have demonstrated previously that pharmacological activation of the CaR stimulates insulin secretion from islet beta-cells and insulin-secreting MIN6 cells. METHODS: In the present study we have investigated the effects of CaR activation on MIN6 cell proliferation and have used shRNA-mediated CaR knockdown to determine whether the CaR is involved in the regulation of insulin secretion via cell-cell communication. RESULTS: CaR activation caused the phosphorylation and activation of the p42/44 MAPK signalling cascade, and this activation was prevented by the shRNA-induced down-regulation of CaR mRNA expression. CaR activation also resulted in increased proliferation of MIN6 cells, consistent with the known role of the p42/44 MAPK system in the regulation of beta-cell proliferation. Down-regulation of CaR expression had no detectable effects on glucose-induced insulin secretion from MIN6 cells maintained as monolayers, but blocked the increases in insulin secretion that were observed when the cells were configured as three-dimensional islet-like structures (pseudoislets), consistent with a role for the CaR in cell-cell communication in pseudoislets. CONCLUSION: It is well established that islet function is dependent on communication between islet cells and the results of this study suggest that the CaR is required for beta-cell to beta-cell interactions within islet-like structures.

Expression and function of the extracellular calcium-sensing receptor in pancreatic beta-cells.

The extracellular calcium-sensing receptor (CaR) was first identified in tissues involved in systemic Ca2+ homeostasis, where it acts to sense changes in circulating Ca2+. It has since been reported that the CaR is expressed in many tissues that are not associated with Ca2+ homeostasis, including the endocrine cells in pancreatic islets of Langerhans. In the present study we have used an insulin-secreting pancreatic beta-cell line (MIN6) to investigate the expression and function of CaR, using the calcimimetic A568, a CaR agonist that activates the CaR at physiological concentrations of extracellular Ca2+ ([Ca2+]o). Immunocytochemistry, Western blotting and RT-PCR confirmed the expression of CaR in MIN6 cells. CaR activation was associated with rapid and transient increases in [Ca2+]o, which were accompanied by the initiation of a marked but transient insulin secretory response. Stimulation of beta-cell secretory activity had no detectable effect on CaR mRNA levels, but CaR mRNA was markedly reduced by configuring MIN6 cells into islet- like structures. Our data are consistent with an important function for the beta-cell CaR in cell - cell communication within islets to co-ordinate insulin secretory responses.

Generation of insulin-expressing cells from mouse embryonic stem cells.

The therapeutic potential of transplantation of insulin-secreting pancreatic beta-cells has stimulated interest in using pluripotent embryonic stem (ES) cells as a starting material from which to generate insulin secreting cells in vitro. Mature beta-cells are endodermal in origin so most reported differentiation protocols rely on the identification of endoderm-specific markers. However, endoderm development is an early event in embryogenesis that produces cells destined for the gut and associated organs in the embryo, and for the development of extra-embryonic structures such as the yolk sac. We have demonstrated that mouse ES cells readily differentiate into extra-embryonic endoderm in vitro, and that these cell populations express the insulin gene and other functional elements associated with beta-cells. We suggest that the insulin-expressing cells generated in this and other studies are not authentic pancreatic beta-cells, but may be of extra-embryonic endodermal origin.