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Anti-VEGF therapies benefit several cancer types, but drug resistance that limits therapeutic response can emerge. We generated cell lines from anti-VEGF–resistant tumor xenografts to investigate the mechanisms by which resistance develops. Of all tumor cells tested, only A431 (A431-V) epidermoid carcinoma cells developed partial resistance to the VEGF inhibitor aflibercept. Compared with the parental tumors, A431-V tumors secreted greater amounts of IL6 and exhibited higher levels of phospho-STAT3. Notably, combined blockade of IL6 receptor (IL6R) and VEGF resulted in enhanced activity against A431-V tumors. Similarly, inhibition of IL6R enhanced the antitumor effects of aflibercept in DU145 prostate tumor cells that displays high endogenous IL6R activity. In addition, post hoc stratification of data obtained from a clinical trial investigating aflibercept efficacy in ovarian cancer showed poorer survival in patients with high levels of circulating IL6. These results suggest that the activation of the IL6/STAT3 pathway in tumor cells may provide a survival advantage during anti-VEGF treatment, suggesting its utility as a source of response biomarkers and as a therapeutic target to heighten efficacious results. Cancer Res; 76(8); 2327–39. ©2016 AACR.

Endoplasmic reticulum (ER) stress has recently been implicated as a cause for obesity-related insulin resistance; however, what causes ER stress in obesity has remained uncertain. Here, we have tested the hypothesis that macronutrients can cause acute (ER) stress in rat liver. Examined were the effects of intravenously infused glucose and/or lipids on proximal ER stress sensor activation (PERK, eIF2-α, ATF4, Xbox protein 1 (XBP1s)), unfolded protein response (UPR) proteins (GRP78, calnexin, calreticulin, protein disulphide isomerase (PDI), stress kinases (JNK, p38 MAPK) and insulin signaling (insulin/receptor substrate (IRS) 1/2 associated phosphoinositol-3-kinase (PI3K)) in rat liver. Glucose and/or lipid infusions, ranging from 23.8 to 69.5 kJ/4 h (equivalent to between ~17% and ~50% of normal daily energy intake), activated the proximal ER stress sensor PERK and ATF6 increased the protein abundance of calnexin, calreticulin and PDI and increased two GRP78 isoforms. Glucose and glucose plus lipid infusions induced comparable degrees of ER stress, but only infusions containing lipid activated stress kinases (JNK and p38 MAPK) and inhibited insulin signaling (PI3K). In summary, physiologic amounts of both glucose and lipids acutely increased ER stress in livers 12-h fasted rats and dependent on the presence of fat, caused insulin resistance. We conclude that this type of acute ER stress is likely to occur during normal daily nutrient intake.

The liver plays a major role in the early hypometabolic and later hypermetabolic phases after severe burn injury. Proteomic analysis was used to identify altered proteins in liver during these two phases. Sprague-Dawley rats were subjected to a full-thickness dorsal burn injury covering 40% of the total body surface area. Controls consisted of sham-treated animals. Liver tissues were collected on postburn days 1 and 7. The proteomic data show greater production of positive acute phase proteins on day 1 than on day 7. Many antioxidant enzymes were coordinately downregulated on day 1, including the potent biliverdin reductase. These antioxidants were restored and in some cases upregulated on day 7. This opposite trend in the change of antioxidant proteins corroborated our finding of more pronounced oxidative stress on day 1 than on day 7 as measured via protein carbonyl content. The changes of metabolic enzymes on days 1 and 7 were consistent with hypo- and hyper-metabolic states, respectively. Furthermore, a previously unreported decrease in ornithine aminotransferase on day 7 may be a key contributor to the observed increased urinary urea excretion during the hypermetabolic phase. Overall, the many differences in protein expression observed on postburn days 1 and 7 reflect the dissimilar hepatic metabolic patterns during the acute and flow phases following burn injury.

OBJECTIVE—To examine fat biopsy samples from lean insulin-sensitive and obese insulin-resistant nondiabetic individuals for evidence of endoplasmic reticulum (ER) stress. RESEARCH DESIGN AND METHODS—Subcutaneous fat biopsies were obtained from the upper thighs of six lean and six obese nondiabetic subjects. Fat homogenates were used for proteomic (two-dimensional gel and MALDI-TOF/TOF), Western blot, and RT-PCR analysis. RESULTS—Proteomic analysis revealed 19 differentially upregulated proteins in fat of obese subjects. Three of these proteins were the ER stress–related unfolded protein response (UPR) proteins calreticulin, protein disulfide-isomerase A3, and glutathione-S-transferase P. Western blotting revealed upregulation of several other UPR stress–related proteins, including calnexin, a membrane-bound chaperone, and phospho c-jun NH2-terminal kinase (JNK)-1, a downstream effector protein of ER stress. RT-PCR analysis revealed upregulation of the spliced form of X-box binding protein-1s, a potent transcription factor and part of the proximal ER stress sensor inositol-requiring enzyme-1 pathway. CONCLUSIONS—These findings represent the first demonstration of UPR activation in subcutaneous adipose tissue of obese human subjects. As JNK can inhibit insulin action and activate proinflammatory pathways, ER stress activation of JNK may be a link between obesity, insulin resistance, and inflammation.

Cigarette smoking, which exposes the lung to high concentrations of reactive oxidant species (ROS) is the major risk factor for chronic obstructive pulmonary disease (COPD). Recent studies indicate that ROS interfere with protein folding in the endoplasmic reticulum and elicit a compensatory response termed the "unfolded protein response" (UPR). The importance of the UPR lies in its ability to alter expression of a variety of genes involved in antioxidant defense, inflammation, energy metabolism, protein synthesis, apoptosis, and cell cycle regulation. The present study used comparative proteomic technology to test the hypothesis that chronic cigarette smoking induces a UPR in the human lung. Studies were performed on lung tissue samples obtained from three groups of human subjects: nonsmokers, chronic cigarette smokers, and ex-smokers. Proteomes of lung samples from chronic cigarette smokers demonstrated 26 differentially expressed proteins (20 were up-regulated, 5 were down-regulated, and 1 was detected only in the smoking group) compared with nonsmokers. Several UPR proteins were up-regulated in smokers compared with nonsmokers and ex-smokers, including the chaperones, glucose-regulated protein 78 (GRP78) and calreticulin; a foldase, protein disulfide isomerase (PDI); and enzymes involved in antioxidant defense. In cultured human airway epithelial cells, GRP78 and the UPR-regulated basic leucine zipper, transcription factors, ATF4 and Nrf2, which enhance expression of important anti-oxidant genes, increased rapidly (24 h) with cigarette smoke extract. These data indicate that cigarette smoke induces a UPR response in the human lung that is rapid in onset, concentration dependent, and at least partially reversible with smoking cessation. We speculate that activation of a UPR by cigarette smoke may protect the lung from oxidant injury and the development of COPD.

The liver is the major source of proteins used throughout the body for various functions. Upon injury or infection, an acute phase response (APR) is initiated in the liver that is primarily mediated by inflammatory cytokines such as interleukin-1β (IL-1β) and interleukin-6. Among others, the APR is characterized by an altered protein synthetic profile. We used two-dimensional gel electrophoresis to study the dynamics of changes in protein synthesis in hepatocytes exposed to these inflammatory cytokines. Protein profiles were quantified using image analysis and further analyzed using multivariate statistical methods. Our results indicate that IL-1β and IL-6 each induces secreted protein responses with distinct dynamics and dose-dependence. Parallel stimulation by IL-1β and IL-6 results in a protein pattern indistinguishable from the IL-1β pattern, indicating a dominant effect of IL-1β over IL-6 at the doses tested. Multidimensional scaling (MDS) of correlation distances between protein secretion levels revealed two protein pairs that are robustly co-secreted across the various cytokine stimulation conditions, suggesting shared regulatory pathways. Finally, we also used multivariate alternating conditional expectation (MACE) to identify transformation functions that discriminated the cytokine-stimulated and untreated hepatocyte-secreted protein profiles. Our analysis indicates that the expression of neutrophil gelatinase-associated lipocalin (NGAL) was sufficient to discriminate between IL-1β and IL-6 stimulation. The combination of proteomics and multivariate analysis is expected to provide new information on the cellular regulatory networks involved in generating specific cellular responses. © 2005 Wiley Periodicals, Inc.

With the importance of mouse as a model to study human diseases and the human and rat plasma/serum two-dimensional (2-D) maps being extensively annotated, this study was aimed at constructing a detailed mouse serum 2-D map. Serum proteins from two different inbred strains of mice (BALB/cJ and C57BL/6J) and mice subjected to two different inflammatory stimuli (20% burn injury and lipopolysaccharide (LPS) injection) were separated on overlapping gels covering pH 3-8 and stained with SYPRO Ruby dye. The tryptic peptides from the resolved spots were analyzed by mass spectrometry, leading to the identification of 38 different gene products. With the exception of major urinary proteins found in abundance in male C57BL/6J mice, little strain difference of the mouse serum 2-D was observed. Many proteins detected in the mouse serum 2-D map were not reported in human or rat serum 2-D maps including epidermal growth factor receptor. Three major murine acute-phase proteins (APPs), haptoglobin, serum amyloid A, and serum amyloid P, were highly induced by both inflammatory stimuli. Image analysis shows that the variations of APPs between these two inflammatory models were not uniform although LPS (100 microg/animal) in general was more effective than 20% burn injury in inducing APPs. Serum amyloid A, much more sensitive to endotoxin than burn injury, may represent a sensitive marker to differentiate these two different inflammatory states.

We examined the role of the pleiotropic cytokine interferon-gamma (IFN-gamma) in initiating the burn injury-induced acute phase response (APR). Two-dimensional (2-D) electrophoresis was used to obtain serum protein profiles from wild-type (WT) and IFN-gamma knockout mice following sham-burn or 20% burn injury. Serum 2-D images from both groups of burn-injured mice were characterized by the upregulation of a similar panel of protein spots. These included the three major murine acute phase proteins haptoglobin, serum amyloid A, and serum amyloid P, that were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. Furthermore, the changes in the levels of these protein spots were very similar between these two groups of mice, as determined by image analysis. Other features of burn-induced APR such as a decrease in total serum protein concentration, an elevated circulation level of the cytokine interleukin-6 (IL-6), and activation of the IL-6 signal transduction protein STAT3 were also evaluated and found to be similar between wild-type and IFN-gamma knockout mice. These results suggest a dispensable role of IFN-gamma in the induction of the hepatic APR in mice following burn injury.