In cancer the extensive methylation found in the bulk of chromatin is reduced, while the normally unmethylated CpG islands become hypermethylated. Regions of solid tumors are transiently and/or chr...

Epithelial ovarian carcinoma (EOC) patients are usually diagnosed at an advanced stage, characterized by interperitoneal carcinomatosis and production of large volumes of ascites. Vascular endothelial growth factor‐A (VEGF‐A) and its main signaling receptor VEGFR2 (KDR) are coexpressed in primary ovarian tumors, ascitic cells and metastases, suggesting the existence of an autocrine VEGF‐A/KDR loop in EOC cells. In the present study, we examined this possibility and explored the role of this autocrine loop in protecting EOC cells from apoptosis under anchorage free growth conditions (anoikis). We found that 3 different EOC cell lines (Caov3, OVCAR3, SKOV3) express both VEGF‐A and its receptors, including KDR. In these cells, KDR is constitutively phosphorylated and is detected both in the cell plasma membrane and in the nucleus. Treating EOC cells with specific internal inhibitors of KDR kinase activity or a VEGF‐A neutralizing antibody abolished KDR autophosphorylation and resulted in significant increase in apoptosis when cells were grown in single‐cell, anchorage‐free conditions. By contrast, these blocking reagents had no effect on cell viability when EOC cells were grown in adhesive monolayers. In summary, our results indicate that an autocrine VEGF‐A/KDR loop exists in EOC cells and that it plays a role in protecting the cells from anoikis. Our results imply that treating EOC patients with VEGF blocking agents may potentially reduce peritoneal dissemination by decreasing vascular permeability as well as inducing apoptosis of shed ovarian cancer cells in ascites.

Cancer is the leading cause of death in dogs, yet there are no established screening paradigms for early detection. Liquid biopsy methods that interrogate cancer-derived genomic alterations in cell-free DNA in blood are being adopted for multi-cancer early detection in human medicine and are now available for veterinary use. The CANcer Detection in Dogs (CANDiD) study is an international, multi-center clinical study designed to validate the performance of a novel multi-cancer early detection “liquid biopsy” test developed for noninvasive detection and characterization of cancer in dogs using next-generation sequencing (NGS) of blood-derived DNA; study results are reported here. In total, 1,358 cancer-diagnosed and presumably cancer-free dogs were enrolled in the study, representing the range of breeds, weights, ages, and cancer types seen in routine clinical practice; 1,100 subjects met inclusion criteria for analysis and were used in the validation of the test. Overall, the liquid biopsy test demonstrated a 54.7% (95% CI: 49.3–60.0%) sensitivity and a 98.5% (95% CI: 97.0–99.3%) specificity. For three of the most aggressive canine cancers (lymphoma, hemangiosarcoma, osteosarcoma), the detection rate was 85.4% (95% CI: 78.4–90.9%); and for eight of the most common canine cancers (lymphoma, hemangiosarcoma, osteosarcoma, soft tissue sarcoma, mast cell tumor, mammary gland carcinoma, anal sac adenocarcinoma, malignant melanoma), the detection rate was 61.9% (95% CI: 55.3–68.1%). The test detected cancer signal in patients representing 30 distinct cancer types and provided a Cancer Signal Origin prediction for a subset of patients with hematological malignancies. Furthermore, the test accurately detected cancer signal in four presumably cancer-free subjects before the onset of clinical signs, further supporting the utility of liquid biopsy as an early detection test. Taken together, these findings demonstrate that NGS-based liquid biopsy can offer a novel option for noninvasive multi-cancer detection in dogs.

Folate and its synthetic form, folic acid (FA), are essential vitamins for the regeneration of S-adenosyl methionine molecules, thereby maintaining adequate cellular methylation. The deregulation of DNA methylation is a contributing factor to carcinogenesis, as alterations in genetic methylation may contribute to stem cell reprogramming and dedifferentiation processes that lead to a cancer stem cell (CSC) phenotype. Here, we investigate the potential effects of FA exposure on DNA methylation and colonosphere formation in cultured human colorectal cancer (CRC) cell lines. We show for the first time that HCT116, LS174T, and SW480 cells grown without adequate FA demonstrate significantly impaired colonosphere forming ability with limited changes in CD133, CD166, and EpCAM surface expression. These differences were accompanied by concomitant changes to DNA methyltransferase (DNMT) enzyme expression and DNA methylation levels, which varied depending on cell line. Taken together, these results demonstrate an interaction between FA metabolism and CSC phenotype in vitro and help elucidate a connection between supplemental FA intake and CRC development.

Epigenetic modifications are involved in the initiation and progression of cancer. Expression patterns and activity of DNA methyltransferases (DNMTs) are strictly controlled in normal cells, however, regulation of these enzymes is lost in cancer cells due to unknown reasons. Cancer therapies which target DNMTs are promising treatments of hematologic cancers, but they lack effectiveness in solid tumors. Solid tumors exhibit areas of hypoxia and hypoglycaemia due to their irregular and dysfunctional vasculature, and we previously showed that hypoxia reduces global DNA methylation. Colorectal carcinoma (CRC) cells (HCT116 and 379.2; p53+/+ and p53-/-, respectively) were subjected to ischemia (hypoxia and hypoglycaemia) in vitro, and levels of DNMTs were assessed. We found a significant decrease in mRNA for DNMT1, DNMT3a and DNMT3b, and similar reductions in DNMT1 and DNMT3a protein levels were detected by western blotting. In addition, total activity levels of DNMTs (as measured by an ELISA-based DNMT activity assay) were reduced in cells exposed to hypoxic and hypoglycaemic conditions. Immunofluorescence of HCT116 tumor xenografts demonstrated an inverse relationship between ischemia (as revealed by carbonic anhydrase IX staining) and DNMT1 protein. Bisulfite sequencing of the proximal promoter region of p16INK4a showed a decrease in 5-methylcytosine following in vitro exposure to ischemia. These studies provide evidence for the down-regulation of DNMTs and modulation of methylation patterns by hypoxia and hypoglycaemia in human CRC cells, both in vitro and in vivo. Our findings suggest that ischemia, either intrinsic or induced through the use of anti-angiogenic drugs, may influence epigenetic patterning and hence tumor progression.