Downstream Signaling Components Research Articles

Regulation of versican expression by bacterial infection is TLR4‐dependent but MyD88‐independent (1046.3)

In the acute response to bacterial lung infection, gram‐negative bacteria activation of toll‐like receptors (TLRs) stimulates macrophages to secrete a variety of molecules that lead initially to pulmonary inflammation and, ultimately, to resolution of inflammation. Using immunohistochemistry and qPCR we have shown that versican, a chondroitin sulfate proteoglycan, co‐localizes with macrophages in the lungs of mice with gram‐negative pneumonia, and is produced by alveolar and bone marrow‐derived macrophages (BMDM) exposed to live E. coli or lipopolysaccharide (LPS) in vitro. The goal of the current study is to define the TLR signaling pathway responsible for versican production by macrophages. BMDM were isolated from wild‐type mice (WT) and mice lacking TLR2 (TLR2 KO), TLR4 (TLR4 KO), or MyD88 (MyD88 KO). Versican mRNA was detected at low levels in untreated BMDM from all mice. In WT BMDM, versican mRNA was significantly increased by 4 h after treatment with 10 ng/ml LPS (50.5 ± 10.8‐fold vs PBS control; n = 3, p < 0.0001), and returned to basal levels by 24 h. The versican response to LPS was similarly elevated in TLR2 KO BMDM but was completely abrogated in TLR4 KO BMDM, indicating that LPS induction of versican is mediated by TLR4. Further, the response in MyD88 KO BMDM (48.3 ± 5.0‐fold vs PBS control; n = 3, p < 0.0001) was similar to that in WT BMDM, indicating that LPS induction of versican is MyD88‐independent. Further studies to define downstream signaling components in more detail are in process and will contribute to our understanding of the pro‐ vs anti‐inflammatory role of versican in the acute response to bacterial infection.Grant Funding Source: Supported by HL098067 & RR030249

Heme Oxygenase System and Hypertension: A Comprehensive Insight

Hypertension is a complex interplay of interrelated etiologies, and the leading risk factor for many cardiovascular morbidity and mortality worldwide. Cardinal pathophysiological features of hypertension include enhanced vascular inflammation, vascular remodeling, vascular contractility and increased oxidative stress. In response to oxidative, inflammatory or other noxious stimuli, many physiological pathways like the heme oxygenase (HO) system are activated in an attempt to counteract tissue insults. However, the pathophysiological activation of the HO system only results to a transient increase of HO activity that fall below the necessary threshold capable of activating the downstream signaling components of the HO system like the soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP) secondary messenger system. Therefore, a more robust potentiation of the HO system by pharmacological agents such as hemin, heme-arginate, cobalt protoporphyrin or through retroviral HO-1 gene delivery would be needed to surmount the threshold for cytoprotection. The HO system modulates cellular homeostasis. Importantly, the HO system plays a vital role in a wide spectrum of physiologic including the regulation of blood vessel tone. Alterations in the activity and expression of HO has been correlated to pathophysiology of hypertension and related complications such as hypertrophy, myocardial infarction and heart failure. Moreover, the cytoprotection exerted by HO is attributable to its catabolic products namely, carbon monoxide, bilirubin/biliverdin, and ferritin that are known to modulate immune, inflammatory and oxidative insults. The growing incidence of hypertension and associated cardiometabolic complications has prompted the need for the exploration of alternative therapeutic strategies like substances capable of potentiating the HO system. This review briefly, highlights the functional significance of the HO system and its downstream signaling molecules including bilirubin/biliverdin, carbon monoxide and ferritin as potential therapeutic modalities for the management of hypertension and its related co-morbid conditions.

Resistin in Dairy Cows: Plasma Concentrations during Early Lactation, Expression and Potential Role in Adipose Tissue

Resistin is an adipokine that has been implicated in energy metabolism regulation in rodents but has been little studied in dairy cows. We determined plasma resistin concentrations in early lactation in dairy cows and investigated the levels of resistin mRNA and protein in adipose tissue and the phosphorylation of several components of insulin signaling pathways one week post partum (1 WPP) and at five months of gestation (5 MG). We detected resistin in mature bovine adipocytes and investigated the effect of recombinant bovine resistin on lipolysis in bovine adipose tissue explants. ELISA showed that plasma resistin concentration was low before calving, subsequently increasing and reaching a peak at 1 WPP, decreasing steadily thereafter to reach pre-calving levels at 6 WPP. Plasma resistin concentration was significantly positively correlated with plasma non esterified fatty acid (NEFA) levels and negatively with milk yield, dry matter intake and energy balance between WPP1 to WPP22. We showed, by quantitative RT-PCR and western blotting, that resistin mRNA and protein levels in adipose tissue were higher at WPP1 than at 5 MG. The level of phosphorylation of several early and downstream insulin signaling components (IRβ, IRS-1, IRS-2, Akt, MAPK ERK1/2, P70S6K and S6) in adipose tissue was also lower at 1 WPP than at 5 MG. Finally, we showed that recombinant bovine resistin increased the release of glycerol and mRNA levels for ATGL (adipose triglyceride lipase) and HSL (hormone-sensitive lipase) in adipose tissue explants. Overall, resistin levels were high in the plasma and adipose tissue and were positively correlated with NEFA levels after calving. Resistin is expressed in bovine mature adipocytes and promotes lipid mobilization in adipose explants in vitro.

Abscisic Acid Regulates Early Seed Development in Arabidopsis by ABI5-Mediated Transcription of SHORT HYPOCOTYL UNDER BLUE1

Seed development includes an early stage of endosperm proliferation and a late stage of embryo growth at the expense of the endosperm in Arabidopsis thaliana. Abscisic acid (ABA) has known functions during late seed development, but its roles in early seed development remain elusive. In this study, we report that ABA-deficient mutants produced seeds with increased size, mass, and embryo cell number but delayed endosperm cellularization. ABSCISIC ACID DEFICIENT2 (ABA2) encodes a unique short-chain dehydrogenase/reductase that functions in ABA biosynthesis, and its expression pattern overlaps that of SHORT HYPOCOTYL UNDER BLUE1 (SHB1) during seed development. SHB1 RNA accumulation was significantly upregulated in the aba2-1 mutant and was downregulated by the application of exogenous ABA. Furthermore, RNA accumulation of the basic/region leucine zipper transcription factor ABSCISIC ACID-INSENSITIVE5 (ABI5), involved in ABA signaling, was decreased in aba2-1. Consistent with this, seed size was also increased in abi5. We further show that ABI5 directly binds to two discrete regions in the SHB1 promoter. Our results suggest that ABA negatively regulates SHB1 expression, at least in part, through the action of its downstream signaling component ABI5. Our findings provide insights into the molecular mechanisms by which ABA regulates early seed development.

Characterization and Structure of the Vaccinia Virus NF-κB Antagonist A46

Successful vaccinia virus (VACV) replication in the host requires expression of viral proteins that interfere with host immunity, such as antagonists of the activation of the proinflammatory transcription factor NF-κB. Two such VACV proteins are A46 and A52. A46 interacts with the Toll-like receptor/interleukin-1R (TIR) domain of Toll-like receptors and intracellular adaptors such as MAL (MyD88 adapter-like), TRAM (TIR domain-containing adapter-inducing interferon-β (TRIF)-related adaptor molecule), TRIF, and MyD88, whereas A52 binds to the downstream signaling components TRAF6 and IRAK2. Here, we characterize A46 biochemically, determine by microscale thermophoresis binding constants for the interaction of A46 with the TIR domains of MyD88 and MAL, and present the 2.0 Å resolution crystal structure of A46 residues 87-229. Full-length A46 behaves as a tetramer; variants lacking the N-terminal 80 residues are dimeric. Nevertheless, both bind to the Toll-like receptor domains of MAL and MyD88 with KD values in the low μm range. Like A52, A46 also shows a Bcl-2-like fold but with biologically relevant differences from that of A52. Thus, A46 uses helices α4 and α6 to dimerize, compared with the α1-α6 face used by A52 and other Bcl-2 like VACV proteins. Furthermore, the loop between A46 helices α4-α5 is flexible and shorter than in A52; there is also evidence for an intramolecular disulfide bridge between consecutive cysteine residues. We used molecular docking to propose how A46 interacts with the BB loop of the TRAM TIR domain. Comparisons of A46 and A52 exemplify how subtle changes in viral proteins with the same fold lead to crucial differences in biological activity.

Ribosomal Protein S6, a Target of Rapamycin, Is Involved in the Regulation of rRNA Genes by Possible Epigenetic Changes in Arabidopsis

The target of rapamycin (TOR) kinase pathway regulates various biological processes, including translation, synthesis of ribosomal proteins, and transcription of rRNA. The ribosomal protein S6 (RPS6) is one of the well known downstream components of the TOR pathway. Ribosomal proteins have been known to have diverse functions in regulating cellular metabolism as well as protein synthesis. So far, however, little is known about other possible role(s) of RPS6 in plants, besides being a component of the 40 S ribosomal subunit and acting as a target of TOR. Here, we report that RPS6 may have a novel function via interaction with histone deacetylase 2B (AtHD2B) that belongs to the plant-specific histone deacetylase HD2 family. RPS6 and AtHD2B were localized to the nucleolus. Co-expression of RPS6 and AtHD2B caused a change in the location of both RPS6 and AtHD2B to one or several nucleolar spots. ChIP analysis suggests that RPS6 directly interacts with the rRNA gene promoter. Protoplasts overexpressing both AtHD2B and RPS6 exhibited down-regulation of pre-18 S rRNA synthesis with a concomitant decrease in transcription of some of the ribosomal proteins, suggesting their direct role in ribosome biogenesis and plant development. This is consistent with the mutation in rps6b that results in reduction in 18 S rRNA transcription and decreased root growth. We propose that the interaction between RPS6 and AtHD2B brings about a change in the chromatin structure of rDNA and thus plays an important role in linking TOR signaling to rDNA transcription and ribosome biogenesis in plants.

Mitogen-Induced B-Cell Proliferation Activates Chk2-Dependent G1/S Cell Cycle Arrest

B-cell activation and proliferation can be induced by a variety of extracellular stimuli. The fate of an activated B cell following mitogen stimulation can be dictated by the strength or duration of the signal, the expression of downstream signaling components necessary to promote proliferation, and the cell intrinsic sensors and regulators of the proliferative program. Previously we have identified the DNA damage response (DDR) signaling pathway as a cell intrinsic sensor that is activated upon latent infection of primary human B cells by Epstein-Barr virus (EBV). Here we have assessed the role of the DDR as a limiting factor in the proliferative response to non-viral B-cell mitogens. We report that TLR9 activation through CpG-rich oligonucleotides induced B-cell hyper-proliferation and an ATM/Chk2 downstream signaling pathway. However, B-cell activation through the CD40 pathway coupled with interleukin-4 (IL-4) promoted proliferation less robustly and only a modest DDR. These two mitogens, but not EBV, modestly induced intrinsic apoptosis that was independent from the DDR. However, all three mitogens triggered a DDR-dependent G1/S phase cell cycle arrest preventing B-cell proliferation. The extent of G1/S arrest, as evidenced by release through Chk2 inhibition, correlated with B-cell proliferation rates. These findings have implications for the regulation of extra-follicular B-cell activation as it may pertain to the development of auto-immune diseases or lymphoma.

Glycyrrhizin inhibits lipopolysaccharide-induced inflammatory response by reducing TLR4 recruitment into lipid rafts in RAW264.7 cells

BackgroundThe aim of this study was to investigate the effect of glycyrrhizin on LPS-induced endotoxemia in mice and clarify the possible mechanism. MethodsAn LPS-induced endotoxemia mouse model was used to confirm the anti-inflammatory activity of glycyrrhizin in vivo. In vitro, RAW264.7 cells were stimulated with LPS in the presence or absence of glycyrrhizin. The expression of cytokines was determined by ELISA. Toll-like receptor 4 (TLR4) was determined by Western blot analysis. Nuclear factor-kB (NF-κB) and Interferon regulatory factor 3 (IRF3) activation were detected by Western blotting and luciferase assay. Lipid raft staining was detected by immunocytochemistry. ResultsIn vivo, the results showed that glycyrrhizin can improve survival during lethal endotoxemia. In vitro, glycyrrhizin dose-dependently inhibited the expression of TNF-α, IL-6, IL-1β and RANTES in LPS-stimulated RAW264.7 cells. Western blot analysis showed that glycyrrhizin suppressed LPS-induced NF-κB and IRF3 activation. However, glycyrrhizin did not inhibit NF-κB and IRF3 activation induced by MyD88-dependent (MyD88, IKKβ) or TRIF-dependent (TRIF, TBK1) downstream signaling components. Moreover, glycyrrhizin did not affect the expression of TLR4 and CD14 induced by LPS. Significantly, we found that glycyrrhizin decreased the levels of cholesterol of lipid rafts and inhibited translocation of TLR4 to lipid rafts. Moreover, glycyrrhizin activated ABCA1, which could induce cholesterol efflux from lipid rafts. ConclusionGlycyrrhizin exerts an anti-inflammatory property by disrupting lipid rafts and inhibiting translocation of TLR4 to lipid rafts, thereby attenuating LPS-mediated inflammatory response. General significanceLearning the anti-inflammatory mechanism of glycyrrhizin is crucial for the anti-inflammatory drug development.

Photochemical Reaction Mechanism of UV-B-Induced Monomerization of UVR8 Dimers as the First Signaling Event in UV-B-Regulated Gene Expression in Plants

The Arabidopsis thaliana UV RESISTANCE LOCUS8 (UVR8) protein has been identified to specifically mediate photomorphogenic UV-B responses by acting as a UV-B photoreceptor. The dimeric structure of the UVR8 protein dissociates into signaling-active monomers upon UV-B exposure, and the monomers rapidly interact with downstream signaling components to regulate gene expression. UVR8 monomers revert to dimers in the absence of UV-B radiation, thereby reversing transcription activation. UVR8 amino acid residues W233 and W285 have been identified to play critical roles in the UVR8 dimer for the response to UV-B irradiation. In the present work, the photoreaction mechanism for UVR8 monomerization is explored with quantum chemical cluster calculations and evaluated by molecular dynamics simulations using the wild-type UVR8 dimer and novel force field parameters developed for intermediate radicals formed in the photochemical process. Three different models are investigated, which show that the preferred mechanism for UVR8 monomerization involves electron transfer from residue W233 to W285 and onward to R338 initiated by UV-B irradiation, coupled to simultaneous proton transfer from W233 to D129 leading to the formation of protonated D129, a deprotonated W233 radical, and a neutral R338 radical. Due to the formation of the neutral R338 radical, salt bridges involving this residue are disrupted together with the concomitant interruption of several other key salt bridges R286-D96, R286-D107, R338-D44, R354-E43, and R354-E53. The resulting large decrease in protein-protein interaction energy arising from this sequence of events leads to the monomerization of the UVR8 dimer. The mechanism presented is in accord with all experimental data available to date.

Intracellular signaling, molecular modeling and docking of human Dectin-1 receptor and its microbial and non-microbial ligands: a novel gateway to control inflammatory and immune diseases

Human Dectin-1 receptor, a type of pattern recognition receptor, is expressed in dendritic cells, monocytes, macrophages and a subset of T-cells. It comprises an extracellular C-type lectin domain (CTLD), transmembrane region and immunoreceptor tyrosine based activation motif (ITAM) containing cytoplasmic domain. Stimulation of CTLD by fungal β-glucans initiates various cellular pro-inflammatory and immune responses that are controlled by downstream signaling components (Syk and Src kinases and PKCδ) of Dectin-1 receptor. Using immunoblotting, FACS analysis, fluorescence microscopy and SPR technique, we confirmed Syk and Src association with Cytopalsmic Domain of Dectin-1 Receptor (CDDR) is dependent on novel down-stream signaling kinase PKCδ expression and activity that directly bind with the Tyr-15 of ITAM motif. Additionally, we have identified osteopontin (OPN) as a non-microbial endogenous ligand that signals through Dectin-1 receptor for activation of NADPH oxidase in human monocytes. It is an acidic secreted glycoprotein highly expressed by immune cells and involved in pathogenesis of atherosclerosis and vascular remodeling. To combat these pathophysiological conditions and drug designing homology model of CTLD, CDDR and OPN have been constructed using bioinformatics tools. The interaction analysis of regulatory molecular complex of Dectin-1, i.e., CDDR and PKCδ is done through protein-protein docking strategy using HADDOCK webserver. OPN and Dectin-1 interaction pattern were also predicted in addition with post translational modification sites: some myristoylation, N-glycosylation sites, tyrosine protein kinases sites and cell attachment sequence. These findings will be a milestone in control of bacterial and fungal infections and intervention of atherosclerosis.

Wnt-11 Signaling in Cardiomyocytes

The Wnt/frizzled signalling pathways play a key role in cardiogenesis and in adult hearts during cardiac remodelling. We investigated whether the expression of Wnt-11 and its anticipated downstream signalling components is altered in human failing hearts and whether Wnt-11 acutely affects Ca2+ transients in adult murine cardiomyocytes.Left ventricles (LV) homogenates of human non-failing hearts (NF, n=4) and end-stage failing hearts with ischemic (ICM, n=5) and dilated (DCM, n=5) cardiomyopathy were used for western blotting with antibodies against Wnt-11, Fzd-4 and Fzd-7 (Wnt receptors) and Dvl-1 (downstream cytoplasmic regulator). Expression of proteins was normalized to GAPDH and is given as a fraction of average protein expression in NF. Ca2+ transients were measured during confocal line scan imaging with Fluo-4 in isolated murine adult cardiomyocytes after 5 (ncells=20) and 25 minutes (ncells=18) incubation with recombinant Wnt-11 (2 µg/ml). Untreated cells served as controls (ncells=27).Wnt-11 expression in LV in ICM (0.85 ± 0.05) and DCM (0.91 ± 0.34) was not significantly changed when compared to NF. However, the expression of Wnt-11 receptors was highly increased: Fzd-4 in ICM (6.25 ± 1.23; p<0.005) and in DCM (4.81 ± 0.82; p<0.005) as well Fzd-7 in ICM (4.48 ± 0.63; p<0.001) and in DCM (4.45 ± 0.76; p<0.001). The expression of Dvl-1 tended to be decreased in ICM (0.63 ± 0.15; p=0.15) and DCM (0.64 ± 0.29; p=0.17). The amplitude of Ca2+ transients (F/F0), time to peak, and time to 50 % decay (RT50) were not altered during 5 and 25 minutes incubation with Wnt-11 when compared to untreated control cells.The expression of the Wnt-receptors, Fzd-4 and Fzd-7 is significantly increased in human end-stage failing hearts, suggesting their regulation in cardiac remodelling and/or dysfunction. Wnt-11 did not acutely alter cardiomyocyte Ca2+ transients in non-diseased cardiomyocytes.

α4β7 Integrin (LPAM-1) is Upregulated at Atherosclerotic Lesions and is Involved in Atherosclerosis Progression

Background/Aims: Integrin activation and lymphocyte migration to the vascular intima is a key event in early atherosclerosis. α4β7 integrin (LPAM-1) and its ligand, mucosal addressin cell adhesion molecule (MAdCAM-1) are known to play an important role in homing of activated lymphocytes to gut-associated lymphoid tissues. However, it is unclear whether α4β7 integrin is involved in the pathogenesis of atherosclerosis. Methods: The expressions of α4β7 integrin and its ligands in atherosclerosis plaques from 12 week high fat diet (HFD) fed ApoE^-/- and C57BL/6 mice were examined using immunofluorescent and immunohistochemical assays, respectively. We also generated ApoE/β7 double deficient mice and compared atherosclerotic lesion development in β7^+/+ApoE^-/- and β7^-/-ApoE^-/- mice that were fed with HFD for 12 weeks. Results: We found an upregulation of α4β7 integrin and its ligands VCAM-1 and MAdCAM-1 at atherosclerosis plaques in Apolipoprotein E deficient (ApoE^-/-) mice fed with HFD for 12 weeks. Over the 12 week HFD period, peripheral blood lymphocyte (PBL) expression of α4β7 integrin increased in parallel with aortic lesion size. A removal of α4β7 integrin by genetic deletion of the β7 chain in the ApoE^-/- mouse resulted in a markedly decreased 12 week-HFD atherosclerotic plaque area. β7^-/- ApoE^-/- macrophages showed reduced acetylated and native LDL uptake and phagocytic activity, revealing possible roles for α4β7 at two distinct stages of macrophage dysfunction during atherogenesis. Finally, a reduced activity of integrin downstream signalling components focal adhesion kinase (FAK) and MAPK/ERK1/2 in macrophage indicates their possible engagement during α4β7 integrin signalling in atherosclerosis. Conclusions: Together our results reveal a critical role of α4β7 in diet-induced atherosclerosis in mouse.

The CBM signalosome: Potential therapeutic target for aggressive lymphoma?

The CBM signalosome plays a pivotal role in mediating antigen-receptor induced NF-κB signaling to regulate lymphocyte functions. The CBM complex forms filamentous structure and recruits downstream signaling components to activate NF-κB. MALT1, the protease component in the CBM complex, cleaves key proteins in the feedback loop of the NF-κB signaling pathway and enhances NF-κB activation. The aberrant activity of the CBM complex has been linked to aggressive lymphoma. Recent years have witnessed dramatic progresses in understanding the assembly mechanism of the CBM complex, and advances in the development of targeted therapy for aggressive lymphoma. Here, we will highlight these progresses and give an outlook on the potential translation of this knowledge from bench to bedside for aggressive lymphoma patients.

Abstract P6-04-12: CDK inhibition mediates tumor suppressive Smad3 action and decreased metastatic phenotypes in triple negative breast cancer cells

Abstract Introduction: Breast cancer onset and disease progression has been linked to members of the TGFb superfamily and their downstream signaling components, the Smads. Changes in Smad signaling have been associated with the dichotomous role of TGFβ in malignancy, mediating both tumor suppressant and tumor promoting behaviors in breast cancer. Our previous work showed that cyclin/CDK mediated phosphorylation of Smad3 resulted in the inhibition of canonical tumor suppressant Smad3 action. We thus hypothesized that activation of CDKs leads to phosphorylation and inhibition of Smad3, releasing cell cycle arrest and promoting cell proliferation and metastasis. Using triple negative breast cancer cell lines, we examined the impact of specific CDK inhibitors (CDKis) on signaling patterns, metastatic phenotypes, and protein expression profiles. Methods: Triple negative cell lines Hs578T, MDA-MB-231 and MDA-MB-436 were treated with CDK2i or CDK4i and Smad3 activity was measured using a luciferase assay. Transwell migration and Matrigel invasion assays were used to show the effect of transfection with Smad3 CDK phosphorylation site mutants, resistant to inhibitory CDK phosphorylation, or treatment with CDK2i or CDK4i on the study cells. Immunoblotting was performed to measure protein expression levels of Smad3, pSmad3 T179, and MMP2. A novel live cell array was implemented to quantify changes in activity of 14 cancer-related transcription factors over a period of 3 days. TUNEL and Ki67 staining analysis and a xenograft mouse model were used to determine impact of CDKis, in combination with paclitaxel chemotherapy, on apoptosis and tumor growth respectively. Results: Treatment of study cells with CDK2i or CDK4i resulted in increased Smad3 activity and decreased migration and invasion in the study cells. Transfection with a 5M Smad3 mutant construct, containing mutations in all 5 CDK phosphorylation sites, resulted in the greatest decrease in cell migration, when compared with cells transfected with both vector control or WT Smad3. CDK2i or CDK4i therapy resulted in decreased Smad3 protein phosphorylation at the T179 site, while total Smad3 levels were unaffected. CDK2i/4i treatment also resulted in decreased MMP2 expression. The array studies revealed decreased activity of EMT transcription factors B-catenin, Snail, Twist, in MDA-MB-231 cells following CDK2i treatment. CDK2i and paclitaxel combination treatment resulted in increased apoptosis and decreased tumor volume and Ki67 staining compared to control treatment. Conclusions: We have shown for the first time that cyclin/CDK activity, in part, mediates the metastasis-associated role of Smad3 in triple negative breast cancer cells. Importantly, both CDK2i and CDK4i treatment resulted in decreased cell migration/invasion and also corresponded to decreased MMP2 expression. Lastly, the array studies revealed CDK2i mediated effects on key transcription factors associated with the promotion of EMT. Future studies will explore the significance of Smad3 interaction with these EMT factors and will pursue the clinical application of CDK2i for patients with triple negative/cyclin overexpressing breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-12.

Key roles for GRB2-associated-binding protein 1, phosphatidylinositol-3-kinase, cyclooxygenase 2, prostaglandin E2 and transforming growth factor alpha in linoleic acid-induced upregulation of lung and breast cancer cell growth

The distribution of omega-6 and omega-3 polyunsaturated fatty acid (PUFA) intake in Western diets is disproportionate, containing an overabundance of the omega-6 PUFA, linoleic acid (LA; C18:2). Increased enrichment with LA has been shown to contribute to the enhancement of tumorigenesis in several cancer models. Previous work has indicated that phosphatidylinositol 3-kinase (PI3K) may play a key role in LA-induced tumorigenesis. However, the modes by which LA affects carcinogenesis have not been fully elucidated. In this study, a mechanism for LA-induced upregulation of cancer cell growth is defined. LA treatment enhanced cellular proliferation in BT-474 human breast ductal carcinoma and A549 human lung adenocarcinoma cell lines. Enrichment of LA increased cyclooxygenase (COX) activity and led to increases in prostaglandin E2 (PGE2), followed by increases in matrix metalloproteinase (MMP) and transforming growth factor alpha (TGF-α) levels, which are all key elements involved in the enhancement of cancer cell growth. Further investigation revealed that LA supplementation in both BT-474 breast and A549 lung cancer cell lines greatly increased the association between the scaffolding protein GRB2-associated-binding protein 1 (Gab1) and epidermal growth factor receptor (EGFR), although Gab1 protein levels were significantly decreased. These LA-induced changes were associated with increases in activated Akt (pAkt), a downstream signaling component in the PI3K pathway. Treatment with inhibitors of EGFR, PI3K and Gab1-specific siRNAs reversed the upregulation of pAkt, as well as the observed increases in cell proliferation by LA in both cell lines. A549 xenograft assessment in athymic nude mice fed high levels of LA exhibited similar increases in EGFR-Gab1 association and increased levels of pAkt, while mice fed with high levels of the omega-3 PUFA, docosahexaenoic acid (DHA; C22:6), demonstrated an opposite response. The involvement of Gab1 in LA-induced tumorigenesis was further defined utilizing murine cell lines that express high levels of Gab1. Significant increases in cell proliferation were observed with the addition of increasing concentrations of LA. However, no changes in cell proliferation were detected in the murine paired cell lines expressing little or no Gab1 protein, establishing Gab1 as major target in LA-induced enhancement of tumorigenesis.

Calcium signaling and the MAPK cascade are required for sperm activation in Caenorhabditis elegans

In nematode, sperm activation (or spermiogenesis), a process in which the symmetric and non-motile spermatids transform into polarized and crawling spermatozoa, is critical for sperm cells to acquire fertilizing competence. SPE-8 dependent and SPE-8 independent pathways function redundantly during sperm activation in both males and hermaphrodites of Caenorhabditis elegans. However, the downstream signaling for both pathways remains unclear. Here we show that calcium signaling and the MAPK cascade are required for both SPE-8 dependent and SPE-8 independent sperm activation, implying that both pathways share common downstream signaling components during sperm activation. We demonstrate that activation of the MAPK cascade is sufficient to activate spermatids derived from either wild-type or spe-8 group mutant males and that activation of the MAPK cascade bypasses the requirement of calcium signal to induce sperm activation, indicating that the MAPK cascade functions downstream of or parallel with the calcium signaling during sperm activation. Interestingly, the persistent activation of MAPK in activated spermatozoa inhibits Major Sperm Protein (MSP)-based cytoskeleton dynamics. We demonstrate that MAPK plays dual roles in promoting pseudopod extension during sperm activation but also blocking the MSP-based, amoeboid motility of the spermatozoa. Thus, though nematode sperm are crawling cells, morphologically distinct from flagellated sperm, and the molecular machinery for motility of amoeboid and flagellated sperm is different, both types of sperm might utilize conserved signaling pathways to modulate sperm maturation.

Abstract C90: Dependence of afatinib and cetuximab resistant lung adenocarcinomas on mTOR signaling.

Abstract Lung adenocarcinomas harboring mutations in the tyrosine kinase domain of the EGFR are sensitive to tyrosine kinase inhibitors (TKIs) erlotinib or gefitinib. Despite a ∼70% response rate to these agents, patients almost inevitably develop resistance on average within a year of starting drug treatment. In 50% of cases, acquired resistance to EGFR TKIs is due to the emergence of a secondary mutation in the tyrosine kinase domain of the receptor (the T790M mutation). Additional less common mechanisms of resistance include activation of bypass signaling pathways via amplification of other receptor tyrosine kinases (RTKs) like MET and HER2 or mutations in genes encoding downstream signaling components such as PIK3CA or BRAF. In rare instances, tumors biopsied at progression show phenotypic transformations such as epithelial-to-mesenchymal transition (EMT) or neuroendocrine differentiation. We previously generated transgenic mice that develop EGFRL858R+T790M-induced lung adenocarcinomas and showed that T790M-mediated resistance could be overcome using a combination of a second generation TKI BIBW-2992 (afatinib) with the anti-EGFR antibody, cetuximab. This preclinical study prompted a Phase IB/II clinical trial testing this drug combination in patients with progressive disease after erlotinib or gefitinib treatment that is showing a promising 30% response rate. Unfortunately patients eventually develop disease progression on this drug combination and the mechanisms of resistance to afatinib+cetuximab are currently unknown. Here, we used an intermittent dosing strategy, previously used to generate erlotinib-resistant tumors in mice, to generate afatinib+cetuximab resistant tumors in xenograft and transgenic mouse models of EGFR mutant lung cancer. In these models, afatinib+cetuximab resistant tumors lacked detectable mutations in the EGFR transgene, the ERBB2 kinase domain or KRAS. Molecular analysis of resistant tumors revealed high levels of activation of the mTOR signaling pathway. Consistent with these findings, two patients with afatinib+cetuximab-resistant tumors exhibited alterations in genes (TSC1 and NF2) that activate the mTOR pathway. In cell culture and in mouse models, such resistance can be overcome by addition of the mTOR pathway inhibitor, rapamycin. These studies are the first to demonstrate mechanisms of acquired resistance to dual inhibition of EGFR in EGFR mutant lung cancer and provide new insight into the biology of this subset of lung cancers with immediate therapeutic implications for patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C90. Citation Format: Valentina Pirazzoli, Caroline Nebhan, Xiaoling Song, Zenta Walter, Guoping Cai, Anna Wurtz, Zhongming Zhao, Elisa Elisa de Stanchina, Leora Horn, David Carbone, Philip J. Stevens, Vincent Miller, Scott Gettinger, William Pao, Katerina Politi. Dependence of afatinib and cetuximab resistant lung adenocarcinomas on mTOR signaling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C90.

Abstract B275: Dovitinib as a single-agent or in combination with other small molecule inhibitors for mutant BRAF melanomas.

Abstract Background: Receptor tyrosine kinases (RTKs) modulate many oncogenic signaling pathways. RTKs can be activated as a result of feedback loops initiated from inhibition of downstream signaling components and contribute to resistance to small molecule targeted therapies. Target-specific inhibitors have provided many advances in cancer treatment, but it is becoming increasingly evident that single-agent treatment approaches are suboptimal. To address this, we performed single-agent compound screens against a panel of melanoma cell lines to select candidate agents for testing in combination agent screens (Cancer Discov 2013 3:52-67). One of the top single-agents effective in mutant BRAF melanomas was dovitinib (TKI258, CHIR258), a multi-RTK inhibitor shown to inhibit numerous type III, IV, and V RTKs. We present data examining the target profile of dovitinib in short-term cultured melanoma cell lines and describe effective partner agents for further testing as combination therapies. Materials and Methods: Twenty-five melanoma cell lines were screened against 150 agents. The most efficacious forty agents from the single-agent screen were collated and three consensus concentrations were determined for each compound to perform pair-wise combinations (Cancer Discov 2013 3:52-67). Growth inhibition was evaluated by ATP luminescent detection by CellTiterGlo®. Colony formation experiments were performed to confirm positive drug interactions. Phosphoproteomic approaches were used to further evaluate targets of dovitinib. Results: Dovitinib was one of the top hits from this single-agent screen that was effective in mutant BRAF melanoma cell lines. Dovitinib does not inhibit mutant BRAF directly, but inhibits RTKs. We found that multiple RTK targets of dovitinib are expressed and active in mutant BRAF melanoma cell lines. Furthermore, we found that dovitinib combines well with vemurafenib and MEK and AKT inhibitors to inhibit colony formation ability and cell proliferation. This was seen in both vemurafenib-sensitive and resistant cell lines. Conclusions: These findings further demonstrate the utility of using both single- and combination agent screening to determine genotype specific compound effects. Furthermore, these results support the rationale for using dovitinib as a partner agent with several small molecule inhibitors for melanoma combination therapy, especially in the vemurafenib-resistant setting. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B275. Citation Format: Casey G. Langdon, Matthew A. Held, Andrew B. Koo, Michael Klein, Zongzhi Liu, James T. Platt, David F. Stern. Dovitinib as a single-agent or in combination with other small molecule inhibitors for mutant BRAF melanomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B275.

Flavor Preferences Conditioned by Oral Monosodium Glutamate in Mice

The prototypic umami substance monosodium glutamate (MSG) reinforces preferences for its own flavor, as well as preferences for flavors associated with it, by conditioning processes. Mice of 3 inbred strains (C57BL/6J (B6), 129P3/J, and FVB/NJ) and 2 taste-knockout (KO) groups derived from the B6 lineage were initially indifferent to 200mM MSG, but this evaluation was altered by forced exposure to MSG. B6 and KO mice acquired an MSG preference, 129 mice remained indifferent, and FVB mice avoided MSG. The shifts in preference imply a postoral basis for MSG effects, suggesting that it could produce preferences for associated flavors. New mice were trained with a conditioned stimulus (CS+) flavor mixed in 200mM MSG and a CS- flavor in water. Similar to the parent B6 strain, mice missing the T1r3 element of an umami receptor or the downstream signaling component Trpm5 learned to prefer the CS+ flavor and subsequently showed similar preferences for MSG in an ascending concentration series. Consistent with their responses to forced exposure, the 129 strain did not acquire a significant CS+ preference, and the FVB strain avoided the CS+ flavor. The 129 and FVB strains showed little attraction in the ascending MSG concentration series. Together, these data indicate that the postoral effects of MSG can modulate responses to its own and MSG-paired flavors. The basis for strain differences in the responses to MSG is not certain, but the taste-signaling elements T1r3 and Trpm5, which are also present in the gut, are not required for mediation of this flavor learning.

Probiotic-fermented purple sweet potato yogurt activates compensatory IGF-IR/PI3K/Akt survival pathways and attenuates cardiac apoptosis in the hearts of spontaneously hypertensive rats

Apoptosis is recognized as a predictor of adverse outcomes in subjects with cardiac diseases. The aim of this study was to explore the effects of probiotic-fermented purple sweet potato yogurt (PSPY) with high γ-aminobutyric acid (GABA) content on cardiac apoptosis in spontaneously hypertensive rat (SHR) hearts. The rats were orally adminsitered with 2different concentrations of PSPY (10 and 100%) or captopril, 15.6mg/kg, body weight (BW)/day. The control group was administered distilled water. DAPI and TUNEL staining were used to detect the numbers of apoptotic cells. A decrease in the number of TUNEL-positive cardiac myocytes was observed in the SHR-PSPY (10 and 100%) groups. In addition, the levels of key components of the Fas receptor- and mitochondrial-dependent apoptotic pathways were determined by western blot analysis. The results revealed that the levels of the key components of the Fas receptor- and mitochondrial-dependent apoptotic pathway were significantly decreased in the SHR-captopril, and 10 and 100% PSPY groups. Additionally, the levels of phosphorylated insulin-like growth factor‑I receptor (p-IGF‑IR) were increased in SHR hearts from the SHR-control group; however, no recovery in the levels of downstream signaling components was observed. In addition, the levels of components of the compensatory IGF-IR-dependent survival pathway (p-PI3K and p-Akt) were all highly enhanced in the left ventricles in the hearts form the SHR-10 and 100% PSPY groups. Therefore, the oral administration of PSPY may attenuate cardiomyocyte apoptosis in SHR hearts by activating IGF‑IR-dependent survival signaling pathways.