Biomarkers of oesophageal neoplasia

Background. Symptomatic oesophageal cancer is usually advanced and terminates fatally. There is a need for biomarkers to help pathologists recognise patients at increased risk of squamous and glandular oesophageal cancers (in the case of Barrett's oesophagus). The work described in this thesis looks at four topics: cytokeratin 7 and 20 phenotypes in Barrett's intestinal metaplasia; the distribution of cardiac, cardio-oxyntic and 'specialised' intestinal-type Barrett's mucosa in relation to glandular dysplasia; dysregulated expression of the proliferation marker Ki67 and the DNA replication-licensing proteins Mcm2 and Mcm5 in glandular and squamous dysplasia; and relates different types of complete (type I) and incomplete (types IIA/IIB) intestinal metaplasia to dysplasia/neoplasia in Barrett's esophagus. Results. Cytokeratin 7 and 20 phenotypes are complex and variable, so likely clinical utility is not immediately obvious. The ubiquitous presence of intestinal metaplasia (IM) in Barrett's mucosa is confirmed, and a very strong association of IM with dysplasia. A positive association exists between dysplasia and type IIB intestinal metaplasia, and an even stronger negative association with type I intestinal metaplasia. This identifies a possibility that robust markers of (complete) small intestinal metaplasia may be associated with low dysplasia risk in Barrett's oesophagus, whereas a potentially colonic phenotype (type IIB IM) is associated with greater risk. Over-expression of the cell-proliferation related antigen Ki67 and the DNA replication licensing proteins Mcm2 and Mcm5 in the differentiated surface compartment of squamous oesophageal epithelium and Barrett's mucosa confirm the Mem proteins are promising markers of oesophageal dysplasia meriting further evaluation, e.g. in the field of non-endoscopic oesophageal cytology

Mitogen-activated protein kinase kinase 5 regulates proliferation and biosynthetic processes in procyclic forms of Trypanosoma brucei

The pathogenic protozoan T. brucei alternates into distinct developmental stages in the mammalian and insect hosts. The mitogen-activated protein kinase (MAPK) signaling pathways transduce extracellular stimuli into a range of cellular responses, which ultimately lead to the adaptation to the external environment. Here, we combined a loss of function approach with stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry (MS) to investigate the role of the mitogen-activated protein kinase kinase 5 (MKK5) in T. brucei. The silencing of MKK5 significantly decreased the proliferation of procyclic forms of T. brucei. To shed light on the molecular alterations associated with this phenotype, we measured the total proteome and phosphoproteome of cells silenced for MKK5. In the total proteome, we observed a general decrease in proteins related to ribosome and translation as well as down-regulation of several components of the fatty acids biosynthesis pathway. In addition, we observed alterations in the protein levels and phosphorylation of key metabolic enzymes, which point toward a suppression of the oxidative metabolism. Taken together, our findings show that the silencing of MKK5 alters cell growth, energy metabolism, protein and fatty acids biosynthesis in procyclic T. brucei

Children’s School-Day Nutrient Intake in Ontario: A Cross-Sectional Observational Study Comparing Students’ Packed Lunches from Two School Schedules

This study compared the caloric and nutrient values of packed lunch contents and consumption in the Balanced School Day (BSD) (two 20 min eating periods) versus the Traditional Schedule (TS) (one 20 min lunch). Foods consumed during school were assessed by direct food observation in 321 grade 3 and 4 students, aged 7–10 years, at 9 BSD and 10 TS elementary schools in Ontario. Packed lunch contents in the BSD were significantly higher than the TS in energy (3128.14 ± 1100.36 vs. 2658.98 ± 951.34 kJ, p \u3c 0.001, respectively). Similarly, carbohydrates, total sugar, protein, fat, saturated fatty acids (SFA), calcium, iron, and sodium were significantly higher in the BSD versus TS packed lunches. Correspondingly, students in the BSD consumed significantly more energy, carbohydrates, total sugar, and SFA compared to the TS. Overall, lunches brought by students in the BSD schedule provided more energy across all macronutrients, with only a few micronutrients showing increased amounts, suggesting two 20 min eating opportunities could contribute to excess caloric intake during school, potentially contributing to the prevalence of childhood overweight and obesity in Canada. Furthermore, packed lunches in both schedules had excess amounts of nutrients of concern and much work is needed to ensure that children in Canada receive nutritious lunches at school

Students’ food intake from home-packed lunches in the traditional versus balanced school day

Purpose: To assess the type and quantity of foods children brought and consumed at school in the balanced school day (BSD), with two 20-minute eating periods, versus the traditional schedule (TS), with one 20-minute lunch. Methods: Direct observation identified food items and amounts in BSD and TS lunches of grade 3 and 4 students (n = 321). Results: The mean (SD) servings of foods packed in BSD lunches were significantly higher than the TS lunches for milk and alternatives (0.69 (0.70) vs 0.47 (0.49), P = 0.02), sugar-sweetened beverages (SSBs; 0.91 (1.24) vs 0.57 (0.99), P = 0.01), and snacks (2.74 (1.55) vs 2.24 (1.48), P \u3c 0.01). Regardless of schedule, only 40.8% of students had vegetables packed in their lunch, whereas 92.8% had snacks. When comparing foods eaten, SSBs and snacks remained significantly higher in the BSD (0.75 (1.02) vs 0.48 (0.83), P = 0.03; 2.37 (1.44) vs 1.93 (1.36), P = 0.01, respectively). The proportion of children (%) whose consumption met one-third of Canada’s Food Guide recommendations for vegetables and fruit was low (27.5% BSD, 31.0% TS). Conclusions: The BSD may have unintended negative consequences on the type and amount of foods packed in school lunches. Support for families should focus on encouraging more vegetables and fruit and fewer SSBs and snacks in packed lunches

Hypoxic cancer–associated fibroblasts increase NCBP2-AS2/HIAR to promote endothelial sprouting through enhanced VEGF signaling

Intratumoral hypoxia causes the formation of dysfunctional blood vessels, which contribute to tumor metastasis and reduce the efficacy of therapeutic treatments. Blood vessels are embedded in the tumor stroma of which cancer-associated fibroblasts (CAFs) constitute a prominent cellular component. We found that hypoxic human mammary CAFs promoted angiogenesis in CAF-endothelial cell cocultures in vitro. Mass spectrometry–based proteomic analysis of the CAF secretome unraveled that hypoxic CAFs contributed to blood vessel abnormalities by altering their secretion of various pro- and anti-angiogenic factors. Hypoxia induced pronounced remodeling of the CAF proteome, including proteins that have not been previously related to this process. Among those, the uncharacterized protein NCBP2-AS2 that we renamed HIAR (hypoxia-induced angiogenesis regulator) was the protein most increased in abundance in hypoxic CAFs. Silencing of HIAR abrogated the pro-angiogenic and pro-migratory function of hypoxic CAFs by decreasing secretion of the pro-angiogenic factor VEGFA and consequently reducing VEGF/VEGFR downstream signaling in the endothelial cells. Our study has identified a regulator of angiogenesis and provides a map of hypoxia-induced molecular alterations in mammary CAFs

Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity

The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion

Proteomics-based metabolic modeling reveals that fatty acid oxidation (FAO) controls endothelial cell (EC) permeability.

Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability

Estimation of gestational age from fundal height: a solution for resource-poor settings

Many women in resource-poor settings lack access to reliable gestational age assessment because they do not know their last menstrual period; there is no ultrasound (US) and methods of newborn gestational age dating are not practised by birth attendants. A bespoke multiple-measures model was developed to predict the expected date of delivery determined by US. The results are compared with both a linear and a nonlinear model. Prospectively collected early US and serial symphysis-pubis fundal height (SFH) data were used in the models. The data were collected from Karen and Burmese women attending antenatal care on the Thai–Burmese border. The multiple-measures model performed best, resulting in a range of accuracy depending on the number of SFH measures recorded per mother (for example six SFH measurements resulted in a prediction accuracy of ±2 weeks). SFH remains the proxy for gestational age in much of the resource-poor world. While more accurate measures should be encouraged, we demonstrate that a formula that incorporates at least three SFH measures from an individual mother and the slopes between them provide a significant increase in the accuracy of prediction compared with the linear and nonlinear formulae also using multiple SFH measures

Colorectal tumors require NUAK1 for protection from oxidative stress

The authors wish to thank the staff of the CRUK Beatson Institute Biological Services Unit for animal husbandry and assistance with in vivo experiments; the staff of the CRUK BI Histology core facility and William Clark of the NGS core facility; David McGarry, Rene Jackstadt, Jiska Van der Reest, Justin Bower and Heather McKinnon for many helpful discussions, and countless colleagues at the CRUK BI and Glasgow Institute of Cancer Sciences for support; Prem Premsrirut & Mirimus Inc. for design and generation of dox-inducible Nuak1 shRNA expressing mice Nathanael Gray for initial provision of NUAK1 inhibitors. Funding was provided by the University of Glasgow and the CRUK Beaton Institute. J.P. was supported by European Commission Marie Curie actions C.I.G. 618448 “SERPLUC” to D.J.M.; N.M. was supported through Worldwide Cancer (formerly AICR) grant 15-0279 to O.J.S. & D.J.M.; B.K. was funded through EC Marie Curie actions mobility award 705190 “NuSiCC”; T.M. was funded through British Lung Foundation grant APHD13-5. The laboratories of S.R.Z. (A12935), O.J.S. (A21139) and M.D. (A17096) are funded by Cancer Research UK. O.J.S. was additionally supported by European Research Council grant 311301 “ColoCan”.Peer reviewedPostprin