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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.

Pharmacologic blockade of the myostatin (Mstn)/activin receptor pathway is being pursued as a potential therapy for several muscle wasting disorders. The functional benefits of blocking this pathway are under investigation, in particular given the findings that greater muscle hypertrophy results from Mstn deficiency arising from genetic ablation compared to post-developmental Mstn blockade. Using high-resolution MS coupled with SILAC mouse technology, we quantitated the relative proteomic changes in gastrocnemius muscle from Mstn knockout (Mstn(-/-) ) and mice treated for 2-weeks with REGN1033, an anti-Mstn antibody. Relative to wild-type animals, Mstn(-/-) mice had a two-fold greater muscle mass and a >1.5-fold change in expression of 12.0% of 1137 quantified muscle proteins. In contrast, mice treated with REGN1033 had minimal changes in muscle proteome (0.7% of 1510 proteins >1.5-fold change, similar to biological difference 0.5% of 1310) even though the treatment induced significant 20% muscle mass increase. Functional annotation of the altered proteins in Mstn(-/-) mice corroborates the mutiple physiological changes including slow-to-fast fiber type switch. Thus, the proteome-wide protein expression differs between Mstn(-/-) mice and mice subjected to specific Mstn blockade post-developmentally, providing molecular-level insights to inform mechanistic hypotheses to explain the observed functional differences.

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.

Burn injury results in immunosuppression; previous work implicated a combination of altered T lymphocyte subpopulations and the elaboration of macrophage-derived mediators. However, the conclusions were based on T cell stimulations in the setting of high-dose polyclonal mitogenic stimuli and a single kinetic time-point. In this study, splenocytes from burned animals were used to examine lymphocyte responses over a multi-day time course following saturating and subsaturating anti-CD3, as well as mixed lymphocyte response (MLR) stimulation. Burn injury resulted in suppressed splenocyte-proliferative responses to high-dose anti-CD3 (2 μg/ml) at all culture time-points (Days 2–5); this inhibition was eliminated by removing macrophages from the splenocyte cultures, by blocking NO production, or by using splenocytes from burned animals congenitally deficient in IFN-γ (IFN-γ−/−). The results are consistent with immunosuppression attributable to burn-induced IFN-γ production, which in turn, drives macrophage NO synthesis (NOS). In MLR cultures, lymphocyte proliferation and IFN-γ production were depressed at later time-points (Days 3–5). APC from burned animals showed no defects as MLR stimulators; T cells from burned animals showed defective, proliferative responses, regardless of the stimulator population. Removing macrophages, adding a NOS inhibitor, or using IFN-γ−/− splenocytes did not restore the MLR response of burned splenocytes. T cells from burned IFN-γ−/− animals also showed depressed proliferation with subsaturating levels of anti-CD3 (0.1 μg/ml); anti-CD-28 augmented the proliferative response. We conclude that burn-induced immunosuppression to authentic antigenic stimulation is related at least in part to defective CD3 signaling pathways and not simply to increased IFN-γ or NO production.

mRNA profiling has been extensively used to study muscle wasting. mRNA level changes may not reflect that of proteins, especially in catabolic muscle where there is decreased synthesis and increased degradation. As sepsis is often associated with burn injury, and burn superimposed by sepsis has been shown to result in significant loss of lean tissues, we characterized changes in the skeletal-muscle proteome of rats subjected to a cutaneous burn covering 20% of the total body surface area, followed 2 days later by sepsis induced by CLP (caecal ligation and puncture). EDL (extensor digitorum longus) muscles were dissected from Burn-CLP animals (n=4) and controls (sham-burned and sham-CLP-treated, n=4). Burn-CLP injury resulted in a rapid loss of EDL weight, increased ubiquitin-conjugated proteins and increased protein carbonyl groups. EDL protein profiles were obtained by two-dimensional gel electrophoresis using two immobilized pH gradient strips with overlapping pH range covering a pH 3–8 range. Seventeen spots were significantly altered in the Burn-CLP compared with the control group, representing 15 different proteins identified by peptide mass fingerprinting. The identities of three proteins including transferrin were further confirmed by liquid chromatography–tandem MS. The significant changes in transferrin and HSP27 (heat-shock protein 27) were verified by Western-blot analysis. HSP60, HSP27 and HSPβ6 were down-regulated, along with HSP70, as detected by Western blotting. Six metabolic enzymes related to energy production were also down-regulated. A simultaneous decrease in chaperone proteins and metabolic enzymes could decrease protein synthesis. Furthermore, decreased HSPs could increase oxidative damage, thus accelerating protein degradation. Using cultured C2C12 myotubes, we showed that H2O2-induced protein degradation in vitro could be partially attenuated by prior heat-shock treatment, consistent with a protective role of HSP70 and/or other HSPs against proteolysis.

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.

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.

Recombinant adeno-associated vectors (rAAV) are commonly purified by either chromatography or equilibrium CsCl gradient. Nevertheless, even after purification various cellular proteins often associate with rAAV vector capsids. Such co-purifying cellular proteins may raise concern about safety of gene therapy. Here we report identification and characterization of the co-purifying cellular protein in the vector preparations by using a combination of two proteomics approaches, GeLC-MS (gel electrophoresis liquid chromatography-mass spectrometry) and 2DE (two-dimensional gel electrophoresis). Most prominent bands revealed by Coomassie Blue staining were mostly similar to the AAV capsid proteins. Posttranslational modifications of capsid proteins were detected by the proteomics analysis. A total of 13 cellular proteins were identified in the rAAV vectors purified by two rounds of cesium chloride gradient centrifugation, including 9 by the GeLC-MS analysis and 4 by the 2DE analysis. Selected cellular proteins were verified by western blot. Furthermore, the cellular proteins could be consistently found associated with different AAV serotypes and carrying different transgenes. Yet, the proteins were not integral components of the viral capsis since a stringent washing procedure by column purification could remove them. These co-purified proteins in AAV vector preparations may have a role in various stages of the AAV life cycle.

Cardiac toxicity is a major limitation in the use of doxorubicin (and related anthracyclins). ON 1910.Na (Estybon, Rogersitib, or 1910), a substituted benzyl styryl sulfone, is equally active as doxorubicin against MCF-7 human mammary carcinoma xenografted into nude mice. 1910 augments the antitumor activity of doxorubicin when given simultaneously. Furthermore, when given in combination, 1910 protects against cardiac weight loss and against morphological damage to cardiac tissues. Doxorubicin induces inactivation of glucose response protein 78 (GRP78), a principal chaperone that serves as the master regulator of the unfolded protein response (UPR). Inactivated GRP78 leads to an increase in misfolded proteins, endoplasmic reticulum (ER) stress, activation of UPR sensors, and increased CHOP expression. 1910 prevents the inactivation of GRP78 by doxorubicin, and the combination, while more active against the tumor, protects against cardiac weight loss.

Embryos produced by somatic cell nuclear transfer (SCNT) display low term developmental potential. This is associated with deficiencies in spindle composition prior to activation and at early mitotic divisions, including failure to assemble certain proteins on the spindle. The protein-deficient spindles are accompanied by chromosome congression defects prior to activation and during the first mitotic divisions of the embryo. The molecular basis for these deficiencies and how they might be avoided are unknown. Proteomic analyses of spindles isolated from normal metaphase II (MII) stage oocytes and SCNT constructs, along with a systematic immunofluorescent survey of known spindle-associated proteins were undertaken. This was the first proteomics study of mammalian oocyte spindles. The study revealed four proteins as being deficient in spindles of SCNT embryos in addition to those previously identified; these were clathrin heavy chain (CLTC), aurora B kinase, dynactin 4, and casein kinase 1 alpha. Due to substantial reduction in CLTC abundance after spindle removal, we undertook functional studies to explore the importance of CLTC in oocyte spindle function and in chromosome congression defects of cloned embryos. Using siRNA knockdown, we demonstrated an essential role for CLTC in chromosome congression during oocyte maturation. We also demonstrated rescue of chromosome congression defects in SCNT embryos at the first mitosis using CLTC mRNA injection. These studies are the first to employ proteomics analyses coupled to functional interventions to rescue a specific molecular defect in cloned embryos.

Oxidative injury is believed to play an important role in the pathogenesis of lung diseases such as emphysema and lung cancer. We examined the effects of a classic reactive oxygen species, H 2O 2, on the hydrogen peroxide response proteins (HPRP) in human pneumocytes using comparative two-dimensional gel electrophoresis (2DE) and peptide mass fingerprinting. Four HPRP-associated proteins (DJ-1, peroxiredoxins [Prxs] I and IV and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) were changed upon exposure to H 2O 2 (1 mM for 24 h). H 2O 2 exposure increased the acid (oxidized) form and decreased the basic (reduced) form of DJ-1 (pI 5.8 and 6.2, respectively), Prx I and IV and GAPDH. Mechanistic studies on DJ-1 indicated that the slow recovery of the reduced form was blocked by cyclohexamide, suggesting that the recovery was due to new protein synthesis. Total DJ-1 expression was decreased by increasing concentrations of H 2O 2. In contrast, a more complex mix of oxidants in the form of cigarette smoke extract (CSE) dose-dependently increased DJ-1 expression and produced a novel DJ-1 isoform (p I 5.6). Moreover, DJ-1 expression was higher in the lungs of chronic cigarette smokers compared with nonsmokers, a result which resembled the effects of CSE in cultured cells. These data indicate that in human pneumocytes, DJ-1 functions as an antioxidant but that no enzymatic system converts the oxidized to the reduced form. Up-regulation of DJ-1 by cigarette smoke may be a compensatory mechanism that protects the lung from oxidative stress-related injury.

Blockade of the VEGF pathway and subsequent inhibition of tumor angiogenesis has been shown to prolong the survival of patients with certain types of solid tumors. However, the extent and duration of the survival benefit is often limited by emergence of resistance. To date, mechanisms of resistance and relevant biomarkers have not been clearly defined. To study mechanisms of anti-VEGF resistance, we used the VEGF inhibitor ziv-aflibercept (also known as VEGF Trap) and derived a resistant tumor line from sensitive A431 human squamous cell carcinomas. A431 tumor cells were implanted subcutaneously into the flank of CB17 SCID mice. When tumors reached approximately 100 -150 mm3, animals were continuously (up to 7 weeks) treated with ziv-aflibercept. Initially, A431 tumors were very responsive to ziv-aflibercept, but resistant outgrowth was observed at 6-7 weeks post treatment start. Outgrowing tumors were harvested, in vivo passaged in the presence of ziv-aflibercept and cell lines were isolated from these resistant tumor variants (A431-V) as well as from in vivo passaged control treated tumors (A431-P). Inherent resistance of A431-V tumors to ziv-aflibercept was confirmed. Using several screening approaches, such as PathScan RTK Signaling Antibody Array, isotope-labeled mass spectrometry and ELISA based assays, the IL-6/STAT3 pathway was identified as one of the most up-regulated signaling pathways in the resistant A431-V model compared to sensitive A431-P cells. IL-6-dependent STAT3 activation was confirmed using the IL-6 receptor blocking antibody sarilumab, which dramatically reduced the constitutive phosphorylation of STAT3 in A431-V cells and tumors. To determine if IL-6 plays a role in anti-VEGF resistance, we assessed the effects of ziv-aflibercept alone or in combination with sarilumab in established subcutaneous A431-V tumors. A431-V tumor resistance to VEGF blockade was abrogated by inhibition of IL-6 signaling; compared to control-treated tumors, combination treatment resulted in 91% tumor growth inhibition 14 days post treatment start, while single agent ziv-aflibercept treatment resulted in 57% tumor growth inhibition. These findings were corroborated in Du145 human prostate tumors with high levels of endogenous IL-6/STAT3 signaling. Combined VEGF and IL-6 blockade showed enhanced anti-tumor activity in DU145 tumor bearing mice compared to single agents. Thus, blockade of the IL-6/STAT3 pathway warrants further investigation in overcoming resistance to anti-VEGF therapy. These data underscore that advanced knowledge of predictive biomarkers as well as resistance mechanisms is crucial for successful patient and treatment selection. Citation Format: Alexander P. Adler, Alexandra Eichten, Li Zhang, Jia Su, Ella Ioffe, George D. Yancopoulus, Douglas MacDonald, Christopher Daly, Xunbao Duan, Gavin Thurston. Evasive resistance to VEGF blockade mediated by autocrine IL-6/STAT3 signaling in xenograft models of human cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1378. doi:10.1158/1538-7445.AM2015-1378