Cancer cells escape autophagy inhibition via NRF2-induced macropinocytosis.

Many cancers, including pancreatic ductal adenocarcinoma (PDAC), depend on autophagy-mediated scavenging and recycling of intracellular macromolecules, suggesting that autophagy blockade should cause tumor starvation and regression. However, until now autophagy-inhibiting monotherapies have not demonstrated potent anti-cancer activity. We now show that autophagy blockade prompts established PDAC to upregulate and utilize an alternative nutrient procurement pathway: macropinocytosis (MP) that allows tumor cells to extract nutrients from extracellular sources and use them for energy generation. The autophagy to MP switch, which may be evolutionarily conserved and not cancer cell restricted, depends on activation of transcription factor NRF2 by the autophagy adaptor p62/SQSTM1. NRF2 activation by oncogenic mutations, hypoxia, and oxidative stress also results in MP upregulation. Inhibition of MP in autophagy-compromised PDAC elicits dramatic metabolic decline and regression of transplanted and autochthonous tumors, suggesting the therapeutic promise of combining autophagy and MP inhibitors in the clinic.

Fructose stimulated de novo lipogenesis is promoted by inflammation.

Benign hepatosteatosis, affected by lipid uptake, de novo lipogenesis and fatty acid (FA) oxidation, progresses to non-alcoholic steatohepatitis (NASH) on stress and inflammation. A key macronutrient proposed to increase hepatosteatosis and NASH risk is fructose. Excessive intake of fructose causes intestinal-barrier deterioration and endotoxaemia. However, how fructose triggers these alterations and their roles in hepatosteatosis and NASH pathogenesis remain unknown. Here we show, using mice, that microbiota-derived Toll-like receptor (TLR) agonists promote hepatosteatosis without affecting fructose-1-phosphate (F1P) and cytosolic acetyl-CoA. Activation of mucosal-regenerative gp130 signalling, administration of the YAP-induced matricellular protein CCN1 or expression of the antimicrobial peptide Reg3b (beta) peptide counteract fructose-induced barrier deterioration, which depends on endoplasmic-reticulum stress and subsequent endotoxaemia. Endotoxin engages TLR4 to trigger TNF production by liver macrophages, thereby inducing lipogenic enzymes that convert F1P and acetyl-CoA to FA in both mouse and human hepatocytes.

The Fire within: Cell-Autonomous Mechanisms in Inflammation-Driven Cancer.

Inflammatory cells are important for tumor initiation and promotion, providing cancer cells with cytokines that enhance cell proliferation and survival. Although malignant epithelial cells were traditionally considered to be on the receiving end of these microenvironmental interactions, recent studies show that epithelial cells can undergo inflammatory reprogramming on their own. Such epigenetic switches are often triggered by chronic tissue injury and play important roles in tissue repair. By converting terminally differentiated cells that harbor even a single oncogenic mutation to a less differentiated state with a higher proliferative potential, cell-autonomous inflammation is an important contributor to tumor initiation.

Stress-Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas.

Despite expression of oncogenic KRAS, premalignant pancreatic intraepithelial neoplasia 1 (PanIN1) lesions rarely become fully malignant pancreatic ductal adenocarcinoma (PDAC). The molecular mechanisms through which established risk factors, such as chronic pancreatitis, acinar cell damage, and/or defective autophagy increase the likelihood of PDAC development are poorly understood. We show that accumulation of the autophagy substrate p62/SQSTM1 in stressed KrasG12D acinar cells is associated with PDAC development and maintenance of malignancy in human cells and mice. p62 accumulation promotes neoplastic progression by controlling the NRF2-mediated induction of MDM2, which acts through p53-dependent and -independent mechanisms to abrogate checkpoints that prevent conversion of differentiated acinar cells to proliferative ductal progenitors. MDM2 targeting may be useful for preventing PDAC development in high-risk individuals.

Hepatic Cholesterol-25-Hydroxylase Overexpression Improves Systemic Insulin Sensitivity in Mice.

Obesity is a major risk factor for several diseases including diabetes, heart disease, and some forms of cancer and due to its rapidly increasing prevalence it has become one of the biggest problems medicine is facing today. All the more surprising, a substantial percentage of obese patients are metabolically healthy when classified based on insulin resistance and systemic inflammation. Oxysterols are naturally occurring molecules that play important role in various metabolic and inflammatory processes and their levels are elevated in patients suffering from obesity and diabetes. 25-Hydroxycholesterol (25-OHC) is produced in cells from cholesterol by the enzyme cholesterol 25-hydroxylase (Ch25h) and is involved in lipid metabolism, inflammatory processes, and cell proliferation. Here, we investigated the role of hepatic Ch25h in the transition from metabolically healthy obesity to insulin resistance and diabetes. Using several different experimental approaches, we demonstrated the significance of Ch25h on the border of "healthy" and "diseased" states of obesity. Adenovirus-mediated Ch25h overexpression in mice improved glucose tolerance and insulin sensitivity and lowered HOMA-IR. Our data suggest that low hepatic Ch25h levels could be considered a risk marker for unhealthy obesity.

Targeting Inflammation in Cancer Prevention and Therapy.

Inflammation is associated with the development and malignant progression of most cancers. As most of the cell types involved in cancer-associated inflammation are genetically stable and thus are not subjected to rapid emergence of drug resistance, the targeting of inflammation represents an attractive strategy both for cancer prevention and for cancer therapy. Tumor-extrinsic inflammation is caused by many factors, including bacterial and viral infections, autoimmune diseases, obesity, tobacco smoking, asbestos exposure, and excessive alcohol consumption, all of which increase cancer risk and stimulate malignant progression. In contrast, cancer-intrinsic or cancer-elicited inflammation can be triggered by cancer-initiating mutations and can contribute to malignant progression through the recruitment and activation of inflammatory cells. Both extrinsic and intrinsic inflammation can result in immunosuppression, thereby providing a preferred background for tumor development. In clinical trials, lifestyle modifications including healthy diet, exercise, alcohol, and smoking cessation have proven effective in ameliorating inflammation and reducing the risk of cancer-related deaths. In addition, consumption of certain anti-inflammatory drugs, including aspirin, can significantly reduce cancer risk, suggesting that common nonsteroidal anti-inflammatory drugs (NSAID) and more specific COX2 inhibitors can be used in cancer prevention. In addition to being examined for their preventative potential, both NSAIDs and more potent anti-inflammatory antibody-based drugs need to be tested for their ability to augment the efficacy of more conventional therapeutic approaches on the basis of tumor resection, radiation, and cytotoxic chemicals. Cancer Prev Res; 9(12); 895-905. ©2016 AACR.

Mass Spectrometry-Based Visualization of Molecules Associated with Human Habitats.

The cars we drive, the homes we live in, the restaurants we visit, and the laboratories and offices we work in are all a part of the modern human habitat. Remarkably, little is known about the diversity of chemicals present in these environments and to what degree molecules from our bodies influence the built environment that surrounds us and vice versa. We therefore set out to visualize the chemical diversity of five built human habitats together with their occupants, to provide a snapshot of the various molecules to which humans are exposed on a daily basis. The molecular inventory was obtained through untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of samples from each human habitat and from the people that occupy those habitats. Mapping MS-derived data onto 3D models of the environments showed that frequently touched surfaces, such as handles (e.g., door, bicycle), resemble the molecular fingerprint of the human skin more closely than other surfaces that are less frequently in direct contact with humans (e.g., wall, bicycle frame). Approximately 50% of the MS/MS spectra detected were shared between people and the environment. Personal care products, plasticizers, cleaning supplies, food, food additives, and even medications that were found to be a part of the human habitat. The annotations indicate that significant transfer of chemicals takes place between us and our built environment. The workflows applied here will lay the foundation for future studies of molecular distributions in medical, forensic, architectural, space exploration, and environmental applications.

Mast cells are not associated with systemic insulin resistance.

BACKGROUND: Infiltration of white adipose tissue (WAT) by inflammatory cells in obesity is considered to be a key event in the development of insulin resistance. Recently, mast cells (MCs) have been identified as new players in the pathogenesis of obesity. We aimed to investigate the relationship between MCs and various inflammatory markers in serum and WAT and to determine the role of MCs in the aetiology of insulin resistance. MATERIALS AND METHODS: Gene expression was measured in WAT from 20 morbidly obese patients and 20 nonobese control subjects. Homoeostasis Model of Assessment-Insulin Resistance (HOMA-IR) was used to estimate insulin sensitivity. In addition, wild-type and mast cell-deficient mice were fed a high-fat or low-fat diet to study mast cell influence on inflammatory cell polarization in WAT and overall metabolic changes. RESULTS: WAT levels of MC-specific TPSb2 transcript were increased in obesity and significantly positively correlated with TNF, CCL2, CCL5 and CD68 gene expression levels in our study subjects after adjustment for sex, age and BMI. Accordingly, MC deficiency abrogated increase in expression of pro-inflammatory M1 macrophage marker genes in mouse WAT upon high-fat diet feeding. However, MCs accumulated in obese human WAT independent of insulin resistance and systemic changes in inflammatory mediators. CONCLUSIONS: Our results suggest that MCs contribute to the local pro-inflammatory state within WAT in obesity but do not play a primary role in causing insulin resistance.

Correction: Circulating Inflammatory Mediators as Potential Prognostic Markers of Human Colorectal Cancer.

[This corrects the article DOI: 10.1371/journal.pone.0148186.].

Circulating Inflammatory Mediators as Potential Prognostic Markers of Human Colorectal Cancer.

BACKGROUND: Cytokines and chemokines in the tumor microenvironment drive metastatic development and their serum levels might mirror the ongoing inflammatory reaction at the tumor site. Novel highly sensitive tools are needed to identify colorectal cancer patients at high risk of recurrence that should be more closely monitored during post-surgical follow up. Here we study whether circulating inflammatory markers might be used to predict recurrence in CRC patients. METHODS: Circulating levels of the inflammatory cytokines IL-1, IL-6, IL-10, TNFalpha, CCL2, CXCL8, VEGF and the acute phase protein Pentraxin-3 were measured by ELISA in preoperative serum samples prospectively collected from a cohort of sixty-nine patients undergoing surgical resection for stage 0-IV CRC and associated with post-operative disease recurrence. RESULTS: Cox multivariate analysis showed that combined high levels (≥ROC cut off-value) of CXCL8, VEGF and Pentraxin3 were associated with increased risk of disease recurrence [HR: 14.28; 95%CI: (3.13-65.1)] independently of TNM staging. Kaplan-Meier analysis showed that CXCL8, VEGF and Pentraxin3 levels were significantly associated with worse survival (P<0.001). CONCLUSIONS: Circulating inflammatory mediators efficiently predicted postoperative recurrence after CRC surgery. Therefore, this study suggest that their validation in large-scale clinical trials may help in tailoring CRC post-surgical management.

Dual prognostic significance of tumour-associated macrophages in human pancreatic adenocarcinoma treated or untreated with chemotherapy.

OBJECTIVE: Tumour-associated macrophages (TAMs) play key roles in tumour progression. Recent evidence suggests that TAMs critically modulate the efficacy of anticancer therapies, raising the prospect of their targeting in human cancer. DESIGN: In a large retrospective cohort study involving 110 patients with pancreatic ductal adenocarcinoma (PDAC), we assessed the density of CD68-TAM immune reactive area (%IRA) at the tumour-stroma interface and addressed their prognostic relevance in relation to postsurgical adjuvant chemotherapy (CTX). In vitro, we dissected the synergism of CTX and TAMs. RESULTS: In human PDAC, TAMs predominantly exhibited an immunoregulatory profile, characterised by expression of scavenger receptors (CD206, CD163) and production of interleukin 10 (IL-10). Surprisingly, while the density of TAMs associated to worse prognosis and distant metastasis, CTX restrained their protumour prognostic significance. High density of TAMs at the tumour-stroma interface positively dictated prognostic responsiveness to CTX independently of T-cell density. Accordingly, in vitro, gemcitabine-treated macrophages became tumoricidal, activating a cytotoxic gene expression programme, inhibiting their protumoural effect and switching to an antitumour phenotype. In patients with human PDAC, neoadjuvant CTX was associated to a decreased density of CD206(+) and IL-10(+) TAMs at the tumour-stroma interface. CONCLUSIONS: Overall, our data highlight TAMs as critical determinants of prognostic responsiveness to CTX and provide clinical and in vitro evidence that CTX overall directly re-educates TAMs to restrain tumour progression. These results suggest that the quantification of TAMs could be exploited to select patients more likely to respond to CTX and provide the basis for novel strategies aimed at re-educating macrophages in the context of CTX.

BAG3 promotes pancreatic ductal adenocarcinoma growth by activating stromal macrophages.

The incidence and death rate of pancreatic ductal adenocarcinoma (PDAC) have increased in recent years, therefore the identification of novel targets for treatment is extremely important. Interactions between cancer and stromal cells are critically involved in tumour formation and development of metastasis. Here we report that PDAC cells secrete BAG3, which binds and activates macrophages, inducing their activation and the secretion of PDAC supporting factors. We also identify IFITM-2 as a BAG3 receptor and show that it signals through PI3K and the p38 MAPK pathways. Finally, we show that the use of an anti-BAG3 antibody results in reduced tumour growth and prevents metastasis formation in three different mouse models. In conclusion, we identify a paracrine loop involved in PDAC growth and metastatic spreading, and show that an anti-BAG3 antibody has therapeutic potential.

Basal autophagy maintains pancreatic acinar cell homeostasis and protein synthesis and prevents ER stress.

Pancreatic acinar cells possess very high protein synthetic rates as they need to produce and secrete large amounts of digestive enzymes. Acinar cell damage and dysfunction cause malnutrition and pancreatitis, and inflammation of the exocrine pancreas that promotes development of pancreatic ductal adenocarcinoma (PDAC), a deadly pancreatic neoplasm. The cellular and molecular mechanisms that maintain acinar cell function and whose dysregulation can lead to tissue damage and chronic pancreatitis are poorly understood. It was suggested that autophagy, the principal cellular degradative pathway, is impaired in pancreatitis, but it is unknown whether impaired autophagy is a cause or a consequence of pancreatitis. To address this question, we generated Atg7(Δpan) mice that lack the essential autophagy-related protein 7 (ATG7) in pancreatic epithelial cells. Atg7(Δpan) mice exhibit severe acinar cell degeneration, leading to pancreatic inflammation and extensive fibrosis. Whereas ATG7 loss leads to the expected decrease in autophagic flux, it also results in endoplasmic reticulum (ER) stress, accumulation of dysfunctional mitochondria, oxidative stress, activation of AMPK, and a marked decrease in protein synthetic capacity that is accompanied by loss of rough ER. Atg7(Δpan) mice also exhibit spontaneous activation of regenerative mechanisms that initiate acinar-to-ductal metaplasia (ADM), a process that replaces damaged acinar cells with duct-like structures.

Heme oxygenase-1 drives metaflammation and insulin resistance in mouse and man.

Obesity and diabetes affect more than half a billion individuals worldwide. Interestingly, the two conditions do not always coincide and the molecular determinants of "healthy" versus "unhealthy" obesity remain ill-defined. Chronic metabolic inflammation (metaflammation) is believed to be pivotal. Here, we tested a hypothesized anti-inflammatory role for heme oxygenase-1 (HO-1) in the development of metabolic disease. Surprisingly, in matched biopsies from "healthy" versus insulin-resistant obese subjects we find HO-1 to be among the strongest positive predictors of metabolic disease in humans. We find that hepatocyte and macrophage conditional HO-1 deletion in mice evokes resistance to diet-induced insulin resistance and inflammation, dramatically reducing secondary disease such as steatosis and liver toxicity. Intriguingly, cellular assays show that HO-1 defines prestimulation thresholds for inflammatory skewing and NF-κB amplification in macrophages and for insulin signaling in hepatocytes. These findings identify HO-1 inhibition as a potential therapeutic strategy for metabolic disease.

Lipoprotein(a) is not related to markers of insulin resistance in pregnancy.

BACKGROUND: Dyslipidemia, a major risk factor for cardiovascular disease is a common finding in patients with type 2 diabetes and among women with gestational diabetes. Elevated levels of lipoprotein(a) [Lp(a)] are linked to increased risk of cardiovascular disease. However, its relationship with insulin resistance, type 2 diabetes and gestational diabetes is controversial and unproven. Here we aimed to clarify whether Lp(a) levels are associated with insulin sensitivity in pregnancy. METHODS: Sixty-four women with gestational diabetes and 165 with normal glucose tolerance were enrolled in the study. Fasting Lp(a) serum levels were measured in all women at 24-28 weeks of gestation. RESULTS: In pregnancy, there was no significant difference in serum Lp(a) concentrations between the two groups. Its level did not correlate with markers of insulin resistance (HOMA-IR), insulin sensitivity (HOMA-S%), pancreatic beta-cell function (HOMA-B%) and insulin sensitivity in dynamic conditions (OGIS). In addition, fasting glucose and insulin levels and those throughout an oral glucose tolerance test were independent of Lp(a) concentrations in our study group. CONCLUSIONS: Lp(a) levels in pregnant women do not differ with respect to the presence or absence of gestational diabetes. Although influenced by some components of the lipid profile, such as triglycerides and HDL-C, insulin resistance in pregnancy is not affected by Lp(a).

Differential geometric analysis of alterations in MH α-helices.

Antigen presenting cells present processed peptides via their major histocompatibility (MH) complex to the T cell receptors (TRs) of T cells. If a peptide is immunogenic, a signaling cascade can be triggered within the T cell. However, the binding of different peptides and/or different TRs to MH is also known to influence the spatial arrangement of the MH α-helices which could itself be an additional level of T cell regulation. In this study, we introduce a new methodology based on differential geometric parameters to describe MH deformations in a detailed and comparable way. For this purpose, we represent MH α-helices by curves. On the basis of these curves, we calculate in a first step the curvature and torsion to describe each α-helix independently. In a second step, we calculate the distribution parameter and the conical curvature of the ruled surface to describe the relative orientation of the two α-helices. On the basis of four different test sets, we show how these differential geometric parameters can be used to describe changes in the spatial arrangement of the MH α-helices for different biological challenges. In the first test set, we illustrate on the basis of all available crystal structures for (TR)/pMH complexes how the binding of TRs influences the MH helices. In the second test set, we show a cross evaluation of different MH alleles with the same peptide and the same MH allele with different peptides. In the third test set, we present the spatial effects of different TRs on the same peptide/MH complex. In the fourth test set, we illustrate how a severe conformational change in an α-helix can be described quantitatively. Taken together, we provide a novel structural methodology to numerically describe subtle and severe alterations in MH α-helices for a broad range of applications.

Inflammation, autophagy, and obesity: common features in the pathogenesis of pancreatitis and pancreatic cancer.

Inflammation and autophagy are cellular defense mechanisms. When these processes are deregulated (deficient or overactivated) they produce pathologic effects, such as oxidative stress, metabolic impairments, and cell death. Unresolved inflammation and disrupted regulation of autophagy are common features of pancreatitis and pancreatic cancer. Furthermore, obesity, a risk factor for pancreatitis and pancreatic cancer, promotes inflammation and inhibits or deregulates autophagy, creating an environment that facilitates the induction and progression of pancreatic diseases. However, little is known about how inflammation, autophagy, and obesity interact to promote exocrine pancreatic disorders. We review the roles of inflammation and autophagy, and their deregulation by obesity, in pancreatic diseases. We discuss the connections among disordered pathways and important areas for future research.

Loss of acinar cell IKKα triggers spontaneous pancreatitis in mice.

Chronic pancreatitis is an inflammatory disease that causes progressive destruction of pancreatic acinar cells and, ultimately, loss of pancreatic function. We investigated the role of IκB kinase α (IKKα) in pancreatic homeostasis. Pancreas-specific ablation of IKKα (Ikkα(Δpan)) caused spontaneous and progressive acinar cell vacuolization and death, interstitial fibrosis, inflammation, and circulatory release of pancreatic enzymes, clinical signs resembling those of human chronic pancreatitis. Loss of pancreatic IKKα causes defective autophagic protein degradation, leading to accumulation of p62-mediated protein aggregates and enhanced oxidative and ER stress in acinar cells, but none of these effects is related to NF-κB. Pancreas-specific p62 ablation prevented ER and oxidative stresses and attenuated pancreatitis in Ikkα(Δpan) mice, suggesting that cellular stress induced by p62 aggregates promotes development of pancreatitis. Importantly, downregulation of IKKα and accumulation of p62 aggregates were also observed in chronic human pancreatitis. Our studies demonstrate that IKKα, which may control autophagic protein degradation through its interaction with ATG16L2, plays a critical role in maintaining pancreatic acinar cell homeostasis, whose dysregulation promotes pancreatitis through p62 aggregate accumulation.

Relationship of pentraxin 3 with insulin sensitivity in gestational diabetes.

BACKGROUND: Pentraxin 3 (PTX3) is a cytokine-inducible molecule expressed in different tissues, the levels of which increase in a response to a variety of inflammatory conditions. Recently, it has been linked to the serum glucose levels and some comorbidities in type 2 diabetes. MATERIALS AND METHODS: Here, we aimed to investigate the role of PTX3 in gestational diabetes mellitus (GDM), which is considered a forerunner of type 2 diabetes. Fasting PTX3 serum levels were measured in 90 women [45 GDM, 45 normal glucose tolerance (NGT)] during pregnancy. In addition, PTX3 was measured during a 2 h, 75 g oral glucose tolerance test (OGTT) in 20 women (10 GDM, 10 NGT) at 24-28 weeks of gestation and in 16 of them after delivery (10GDM, 6 NGT). RESULTS: A continuous increase in PTX3 levels was observed during the OGTT and reached in the GDM group a significant difference after 120 min compared with baseline (P < 0·05). Additionally, a rise in the PTX3 concentration was significantly higher in the GDM- compared with the NGT group, 120 min after glucose challenge (P < 0·01). During pregnancy, serum glucose and C-peptide were positively correlated with the PTX3 levels in the whole study group, whereas a negative association was found with the insulin sensitivity parameters QUICKI and OGIS. CONCLUSIONS: Dependence of PTX3 on serum glucose levels was more pronounced in women with GDM than in the NGT group. This notion together with its inverse relation to the parameters of insulin sensitivity, suggests a potential involvement of PTX3 in GDM pathology.