Tumor matrix stiffness promotes metastatic cancer cell interaction with the endothelium.

Tumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness-induced CCN1 activates ß-catenin nuclear translocation and signaling and that this contributes to upregulate N-cadherin levels on the surface of the endothelium, in vitro This facilitates N-cadherin-dependent cancer cell-endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness-induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis.

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.

Parental Reports of Lunch-Packing Behaviours Lack Accuracy: Reported Barriers and Facilitators to Packing School Lunches.

PURPOSE: Parents influence the foods their children consume and often provide proxy reports of this intake. One way parents exert this influence is by providing home-packed lunches. This study compared parental reports of foods packed in children's lunches with what was actually packed and identified parental barriers and facilitators to packing lunches. METHODS: Grade 3 and 4 student-parent dyads (n = 321) in 19 elementary schools in Ontario participated. Parental reports and actual packed lunch contents were collected via self-administered surveys and direct observation, respectively. Parental barriers and facilitators were obtained through open and closed survey questions. RESULTS: Median portions packed were significantly higher for sugar-sweetened beverages and snacks and significantly lower for fruits, fruit juice, vegetables, milk/alternatives, and meat/alternatives than parents reported. Packing a healthy lunch was "important/very important/of the utmost importance" for 95.9% of respondents, and 97.5% perceived their nutrition knowledge as "adequate/good/very good". Barriers to packing a lunch included: child's food preferences, time, finances, allergy policies, and food safety. Nutrition resources, observing other children's lunches, child's input, and planning ahead were identified as facilitators. CONCLUSIONS: Strategies to improve packed lunches should move beyond parental nutrition knowledge and importance of lunch packing to address parental barriers and facilitators.

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.

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 < 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.

In-Depth Proteomics Identifies a Role for Autophagy in Controlling Reactive Oxygen Species Mediated Endothelial Permeability.

Endothelial cells (ECs) form the inner layer of blood vessels and physically separate the blood from the surrounding tissue. To support tissues with nutrients and oxygen, the endothelial monolayer is semipermeable. When EC permeability is altered, blood vessels are not functional, and this is associated with disease. A comprehensive knowledge of the mechanisms regulating EC permeability is key in developing strategies to target this mechanism in pathologies. Here we have used an in vitro model of human umbilical vein endothelial cells mimicking the formation of a physiologically permeable vessel and performed time-resolved in-depth molecular profiling using stable isotope labeling by amino acids in cell culture mass spectrometry (MS)-proteomics. Autophagy is induced when ECs are assembled into a physiologically permeable monolayer. By using siRNA and drug treatment to block autophagy in combination with functional assays and MS proteomics, we show that ECs require autophagy flux to maintain intracellular reactive oxygen species levels, and this is required to maintain the physiological permeability of the cells.

Quantitative phosphoproteomics unveils temporal dynamics of thrombin signaling in human endothelial cells.

Thrombin is the key serine protease of the coagulation cascade and a potent trigger of protease-activated receptor 1 (PAR1)-mediated platelet aggregation. In recent years, PAR1 has become an appealing target for anticoagulant therapies. However, the inhibitors that have been developed so far increase bleeding risk in patients, likely because they interfere with endogenous PAR1 signaling in the endothelium. Because of its complexity, thrombin-induced signaling in endothelial cells has remained incompletely understood. Here, we have combined stable isotope amino acids in cell culture, affinity-based phosphopeptide enrichment, and high-resolution mass spectrometry and performed a time-resolved analysis of the thrombin-induced signaling in human primary endothelial cells. We identified 2224 thrombin-regulated phosphorylation sites, the majority of which have not been previously related to thrombin. Those sites were localized on proteins that are novel to thrombin signaling, but also on well-known players such as PAR1, Rho-associated kinase 2, phospholipase C, and proteins related to actin cytoskeleton, cell-cell junctions, and Weibel-Palade body release. Our study provides a unique resource of phosphoproteins and phosphorylation sites that may generate novel insights into an intimate understanding of thrombin-mediated PAR signaling and the development of improved PAR1 antagonists that affect platelet but not endothelial cell function.

Cancer-associated fibroblasts produce matrix-bound vesicles that influence endothelial cell function.

Intercellular communication between different cell types in solid tumors contributes to tumor growth and metastatic dissemination. The secretome of cancer-associated fibroblasts (CAFs) plays major roles in these processes. Using human mammary CAFs, we showed that CAFs with a myofibroblast phenotype released extracellular vesicles that transferred proteins to endothelial cells (ECs) that affected their interaction with immune cells. Mass spectrometry-based proteomics identified proteins transferred from CAFs to ECs, which included plasma membrane receptors. Using THY1 as an example of a transferred plasma membrane-bound protein, we showed that CAF-derived proteins increased the adhesion of a monocyte cell line to ECs. CAFs produced high amounts of matrix-bound EVs, which were the primary vehicles of protein transfer. Hence, our work paves the way for future studies that investigate how CAF-derived matrix-bound EVs influence tumor pathology by regulating the function of neighboring cancer, stromal, and immune cells.

Omentum-derived matrix enables the study of metastatic ovarian cancer and stromal cell functions in a physiologically relevant environment.

High-grade serous (HGS) ovarian cancer is the most lethal gynaecological disease in the world and metastases is a major cause. The omentum is the preferential metastatic site in HGS ovarian cancer patients and in vitro models that recapitulate the original environment of this organ at cellular and molecular level are being developed to study basic mechanisms that underpin this disease. The tumour extracellular matrix (ECM) plays active roles in HGS ovarian cancer pathology and response to therapy. However, most of the current in vitro models use matrices of animal origin and that do not recapitulate the complexity of the tumour ECM in patients. Here, we have developed omentum gel (OmGel), a matrix made from tumour-associated omental tissue of HGS ovarian cancer patients that has unprecedented similarity to the ECM of HGS omental tumours and is simple to prepare. When used in 2D and 3D in vitro assays to assess cancer cell functions relevant to metastatic ovarian cancer, OmGel performs as well as or better than the widely use Matrigel and does not induce additional phenotypic changes to ovarian cancer cells. Surprisingly, OmGel promotes pronounced morphological changes in cancer associated fibroblasts (CAFs). These changes were associated with the upregulation of proteins that define subsets of CAFs in tumour patient samples, highlighting the importance of using clinically and physiologically relevant matrices for in vitro studies. Hence, OmGel provides a step forward to study the biology of HGS omental metastasis. Metastasis in the omentum are also typical of other cancer types, particularly gastric cancer, implying the relevance of OmGel to study the biology of other highly lethal cancers.

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 < 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.

Neuropilin 1 and its inhibitory ligand mini-tryptophanyl-tRNA synthetase inversely regulate VE-cadherin turnover and vascular permeability.

The formation of a functional blood vessel network relies on the ability of endothelial cells (ECs) to dynamically rearrange their adhesive contacts in response to blood flow and guidance cues, such as vascular endothelial growth factor-A (VEGF-A) and class 3 semaphorins (SEMA3s). Neuropilin 1 (NRP1) is essential for blood vessel development, independently of its ligands VEGF-A and SEMA3, through poorly understood mechanisms. Grounding on unbiased proteomic analysis, we report here that NRP1 acts as an endocytic chaperone primarily for adhesion receptors on the surface of unstimulated ECs. NRP1 localizes at adherens junctions (AJs) where, interacting with VE-cadherin, promotes its basal internalization-dependent turnover and favors vascular permeability initiated by histamine in both cultured ECs and mice. We identify a splice variant of tryptophanyl-tRNA synthetase (mini-WARS) as an unconventionally secreted extracellular inhibitory ligand of NRP1 that, by stabilizing it at the AJs, slows down both VE-cadherin turnover and histamine-elicited endothelial leakage. Thus, our work shows a role for NRP1 as a major regulator of AJs plasticity and reveals how mini-WARS acts as a physiological NRP1 inhibitory ligand in the control of VE-cadherin endocytic turnover and vascular permeability.

Cancer-associated fibroblasts require proline synthesis by PYCR1 for the deposition of pro-tumorigenic extracellular matrix.

Elevated production of collagen-rich extracellular matrix is a hallmark of cancer-associated fibroblasts (CAFs) and a central driver of cancer aggressiveness. Here we find that proline, a highly abundant amino acid in collagen proteins, is newly synthesized from glutamine in CAFs to make tumour collagen in breast cancer xenografts. PYCR1 is a key enzyme for proline synthesis and highly expressed in the stroma of breast cancer patients and in CAFs. Reducing PYCR1 levels in CAFs is sufficient to reduce tumour collagen production, tumour growth and metastatic spread in vivo and cancer cell proliferation in vitro. Both collagen and glutamine-derived proline synthesis in CAFs are epigenetically upregulated by increased pyruvate dehydrogenase-derived acetyl-CoA levels. PYCR1 is a cancer cell vulnerability and potential target for therapy; therefore, our work provides evidence that targeting PYCR1 may have the additional benefit of halting the production of a pro-tumorigenic extracellular matrix. Our work unveils new roles for CAF metabolism to support pro-tumorigenic collagen production.

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.

Modified classification for adenocarcinoma of the gastro-oesophageal junction.

BACKGROUND: Incidence of the gastro-oesophageal junction adenocarcinoma is increasing. Siewert's classification subdivides junctional adenocarcinomas anatomically. Cytokeratin (CK) 7 and 20 immunophenotypes differentiate Barrett's intestinal metaplasia (IM) from gastric IM. Comparing CK immunostaining with Siewert's classification may establish tumour origin and influence surgical choice. METHODS: In this experimental study, 57 patients with gastro-oesophageal junction adenocarcinoma were subdivided endoscopically into 15 type 1, 26 type 2 and 16 type 3 adenocarcinomas. Representative biopsies were immunostained for CK7 and CK20. RESULTS: Intestinal metaplasia was associated with type 1 adenocarcinoma in 12 of 15 patients, 80%; with type 2 in 13 of 26 patients, 50% and type 3 in 6 of 16 patients, 37.5%. All type 1 patients showed Barrett's CK7/CK20 phenotype within IM; type 2 a mixture: 69% (n=9) Barrett's CK7/CK20 and 31% (n=4) gastric CK7/CK20 whereas type 3 patients had a gastric CK7/CK20 pattern in 83% (n=5). Immunostaining within the adenocarcinoma was variable. CONCLUSION: Siewert's type 1 adenocarcinomas express Barrett's CK7/CK20 pattern, type 3 a gastric CK7/CK20 pattern and type 2 tumours a mixture of Barrett's and gastric CK7/CK20 patterns within associated IM. CK immunostaining may refine Siewert's classification into oesophageal type 1 or gastric type 2 adenocarcinoma with IM.

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.

E-cadherin interactome complexity and robustness resolved by quantitative proteomics.

E-cadherin-mediated cell-cell adhesion and signaling plays an essential role in development and maintenance of healthy epithelial tissues. Adhesiveness mediated by E-cadherin is conferred by its extracellular cadherin domains and is regulated by an assembly of intracellular adaptors and enzymes associated with its cytoplasmic tail. We used proximity biotinylation and quantitative proteomics to identify 561 proteins in the vicinity of the cytoplasmic tail of E-cadherin. In addition, we used proteomics to identify proteins associated with E-cadherin-containing adhesion plaques from a cell-glass interface, which enabled the assignment of cellular localization to putative E-cadherin-interacting proteins. Moreover, by tagging identified proteins with GFP (green fluorescent protein), we determined the subcellular localization of 83 putative E-cadherin-proximal proteins and identified 24 proteins that were previously uncharacterized as part of adherens junctions. We constructed and characterized a comprehensive E-cadherin interaction network of 79 published and 394 previously uncharacterized proteins using a structure-informed database of protein-protein interactions. Finally, we found that calcium chelation, which disrupts the interaction of the extracellular E-cadherin domains, did not disrupt most intracellular protein interactions with E-cadherin, suggesting that the E-cadherin intracellular interactome is predominantly independent of cell-cell adhesion.

Quantitative mass spectrometry-based proteomics in angiogenesis.

The process of new blood vessel formation from pre-existing ones is called angiogenesis. Beyond playing a critical role in the physiological development of the vascular system, angiogenesis is a well-recognised hallmark of cancer. Unbiased system-wide approaches are required to complement the current knowledge, and intimately understand the molecular mechanisms regulating this process in physiological and pathological conditions. In this review we describe the cellular and molecular dynamics regulating the physiological growth of vessels and their deregulation in cancer, survey in vitro and in vivo models currently exploited to investigate various aspects of angiogenesis and describe state-of-the-art and most widespread methods and technologies in MS shotgun proteomics. Finally, we focus on current applications of MS to better understand endothelial cell behaviour and propose how modern proteomics can impact on angiogenesis research.

In vivo SILAC-based proteomics reveals phosphoproteome changes during mouse skin carcinogenesis.

Cancer progresses through distinct stages, and mouse models recapitulating traits of this progression are frequently used to explore genetic, morphological, and pharmacological aspects of tumor development. To complement genomic investigations of this process, we here quantify phosphoproteomic changes in skin cancer development using the SILAC mouse technology coupled to high-resolution mass spectrometry. We distill protein expression signatures from our data that distinguish between skin cancer stages. A distinct phosphoproteome of the two stages of cancer progression is identified that correlates with perturbed cell growth and implicates cell adhesion as a major driver of malignancy. Importantly, integrated analysis of phosphoproteomic data and prediction of kinase activity revealed PAK4-PKC/SRC network to be highly deregulated in SCC but not in papilloma. This detailed molecular picture, both at the proteome and phosphoproteome level, will prove useful for the study of mechanisms of tumor progression.