Inhibitor-Sensitive FGFR1 Amplification in Human Non-Small Cell Lung Cancer

Background Squamous cell lung carcinomas account for approximately 25% of new lung carcinoma cases and 40,000 deaths per year in the United States. Although there are multiple genomically targeted therapies for lung adenocarcinoma, none has yet been reported in squamous cell lung carcinoma. Methodology/Principal Findings Using SNP array analysis, we found that a region of chromosome segment 8p11-12 containing three genes–WHSC1L1, LETM2, and FGFR1–is amplified in 3% of lung adenocarcinomas and 21% of squamous cell lung carcinomas. Furthermore, we demonstrated that a non-small cell lung carcinoma cell line harboring focal amplification of FGFR1 is dependent on FGFR1 activity for cell growth, as treatment of this cell line either with FGFR1-specific shRNAs or with FGFR small molecule enzymatic inhibitors leads to cell growth inhibition. Conclusions/Significance These studies show that FGFR1 amplification is common in squamous cell lung cancer, and that FGFR1 may represent a promising therapeutic target in non-small cell lung cancer.Novartis Pharmaceuticals CorporationAmerican Lung AssociationUniting Against Lung CancerSara Thomas Monopoli FundSeaman FoundationIndia. Dept. of BiotechnologyNational Lung Cancer Partnershi

Leishmania donovani exploits host deubiquitinating enzyme A20, a negative regulator of TLR signaling, to subvert host immune response

TLRs, which form an interface between mammalian host and microbe, play a key role in pathogen recognition and initiation of proinflammatory response thus stimulating antimicrobial activity and host survival. However, certain intracellular pathogens such as Leishmania can successfully manipulate the TLR signaling, thus hijacking the defensive strategies of the host. Despite the presence of lipophosphoglycan, a TLR2 ligand capable of eliciting host-defensive cytokine response, on the surface of Leishmania, the strategies adopted by the parasite to silence the TLR2-mediated proinflammatory response is not understood. In this study, we showed that Leishmania donovani modulates the TLR2-mediated pathway in macrophages through inhibition of the IKK-NF-κB cascade and suppression of IL-12 and TNF-α production. This may be due to impairment of the association of TRAF6 with the TAK-TAB complex, thus inhibiting the recruitment of TRAF6 in TLR2 signaling. L. donovani infection drastically reduced Lys 63-linked ubiquitination of TRAF6, and the deubiquitinating enzyme A20 was found to be significantly upregulated in infected macrophages. Small interfering RNA-mediated silencing of A20 restored the Lys 63-linked ubiquitination of TRAF6 as well as IL-12 and TNF-α levels with a concomitant decrease in IL-10 and TGF-ß synthesis in infected macrophages. Knockdown of A20 led to lower parasite survival within macrophages. Moreover, in vivo silencing of A20 by short hairpin RNA in BALB/c mice led to increased NF-κB DNA binding and host-protective proinflammatory cytokine response resulting in effective parasite clearance. These results suggest that L. donovani might exploit host A20 to inhibit the TLR2-mediated proinflammatory gene expression, thus escaping the immune responses of the host

Uncoupling Protein 2 Negatively Regulates Mitochondrial Reactive Oxygen Species Generation and Induces Phosphatase-Mediated Anti-Inflammatory Response in Experimental Visceral Leishmaniasis

To reside and multiply successfully within the host macrophages, Leishmania parasites impair the generation of reactive oxygen species (ROS), which are a major host defense mechanism against any invading pathogen. Mitochondrial uncoupling proteins are associated with mitochondrial ROS generation, which is the major contributor of total cellular ROS generation. In the present study we have demonstrated that Leishmania donovani infection is associated with strong upregulation of uncoupling protein 2 (UCP2), a negative regulator of mitochondrial ROS generation located at the inner membrane of mitochondria. Functional knockdown of macrophage UCP2 by small interfering RNA-mediated silencing was associated with increased mitochondrial ROS generation, lower parasite survival, and induction of marked proinflammatory cytokine response. Induction of proinflammatory cytokine response in UCP2 knocked-down cells was a direct consequence of p38 and ERK1/2 MAPK activation, which resulted from ROS-mediated inhibition of protein tyrosine phosphatases (PTPs). Administration of ROS quencher, N-acetyl-Lcysteine, abrogated PTP inhibition in UCP2 knocked-down infected cells, implying a role of ROS in inactivating PTP. Short hairpin RNA-mediated in vivo silencing of UCP2 resulted in decreased Src homology 2 domain-containing tyrosine phosphatase 1 and PTP-1B activity and host-protective proinflammatory cytokine response resulting in effective parasite clearance. To our knowledge, this study, for the first time, reveals the induction of host UCP2 expression during Leishmania infection to downregulate mitochondrial ROS generation, thereby possibly preventing ROS-mediated PTP inactivation to suppress macrophage defense mechanisms

Leishmania donovani targets tumor necrosis factor receptor-associated factor (TRAF) 3 for impairing TLR4-mediated host response

Intramacrophage pathogen Leishmania donovani escapes host immune response by subverting Toll-like receptor (TLR) signaling, which is critically regulated by protein ubiquitination. In the present study, we identified tumor necrosis factor receptorassociated factor (TRAF) 3, degradative ubiquitination of which is essential for TLR4 activation, as a target for Leishmania to deactivate LPS-mediated TLR4 signaling. We used LPS-treated RAW 264.7 cells and compared the TLR4-mediated immune response in these cells with L. donovani and L. donovani � LPS costimulated macrophages. TRAF3, which was ubiquitinated (2.1- fold over control) at lys 48 position and subsequently degraded following LPS treatment, persisted in L. donovani and L. donovani � LPS costimulated cells due to defective lys 48 ubiquitination. Lys 63-linked ubiquitinatio of upstream proteins in the cascade (cIAP1/2 and TRAF6), mandatory for TRAF3 degradation, was also reduced postinfection. This may be attributed to reduced association between ubiquitin-conjugating enzyme Ubc13 and TRAF6 during infection. Inhibition of TRAF3 before infection by shRNA in Balb/c mice showed enhanced IL-12 and TNF-� (10.8- and 8.1-fold over infected control) and decreased spleen parasite burden (61.3% suppression, P<0.001), thereby marking reduction in disease progression. Our findings identified TRAF3 as a novel molecular regulator exploited by Leishmania for successful infection.—Gupta, P., Giri, J., Srivastav, S., Chande, A. G., Mukhopadhyaya, R., Das, P. K., Ukil, A. Leishmania donovani targets tumor necrosis factor receptor-associated factor (TRAF) 3 for impairing TLR4-mediated host response

Modeling and Experimental Analyses Reveals Signaling Plasticity in a Bi-Modular Assembly of CD40 Receptor Activated Kinases

<div><p>Depending on the strength of signal dose, CD40 receptor (CD40) controls ERK-1/2 and p38MAPK activation. At low signal dose, ERK-1/2 is maximally phosphorylated but p38MAPK is minimally phosphorylated; as the signal dose increases, ERK-1/2 phosphorylation is reduced whereas p38MAPK phosphorylation is reciprocally enhanced. The mechanism of reciprocal activation of these two MAPKs remains un-elucidated. Here, our computational model, coupled to experimental perturbations, shows that the observed reciprocity is a system-level behavior of an assembly of kinases arranged in two modules. Experimental perturbations with kinase inhibitors suggest that a minimum of two trans-modular negative feedback loops are required to reproduce the experimentally observed reciprocity. The bi-modular architecture of the signaling pathways endows the system with an inherent plasticity which is further expressed in the skewing of the CD40-induced productions of IL-10 and IL-12, the respective anti-inflammatory and pro-inflammatory cytokines. Targeting the plasticity of CD40 signaling significantly reduces <em>Leishmania major</em> infection in a susceptible mouse strain. Thus, for the first time, using CD40 signaling as a model, we show how a bi-modular assembly of kinases imposes reciprocity to a receptor signaling. The findings unravel that the signalling plasticity is inherent to a reciprocal system and that the principle can be used for designing a therapy.</p> </div

Experimental perturbation studies by the inhibition of kinases.

<p>(A–F) Mean of three in vivo inhibition studies on the peritoneal macrophages after treatment with anti- CD40 antibody of medium strength (3 µg/ml). Inhibitors of (A) lyn (PP1; 170 nM), (B) PI3-K (Ly294002; 15 µM), (C) p38MAPK (SB203580; 10 µg/ml), (D) Syk (Piceatannol (PC); 20 µM), (E) Raf-1 (Radicicol (Rad); 18 nM), (F)ERK-1/2 (PD098059; 100 µM). The representative western blots are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039898#pone.0039898.s007" target="_blank">figure S7</a> and the densitometric analysis results are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039898#pone.0039898.s011" target="_blank">dataset S1</a>. In the figures, “p-Kinase” represents the phosphorylated kinase. Here “U-In” and “–In” means un-inhibited and inhibited, respectively.</p

Kinetics of reciprocal signal propagation.

<p>The kinetics of phosphorylation of top layer kinases syk, lyn and terminal layer kinases ERK-1/2 and p38MAPK. Ratio of phosphorylation (phosphorylated kinase/Total kinase) of the kinases at 5, 15, 30, 60 minutes is shown for three doses- L, M and H- of αCD40. The representative western bolts are given as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039898#pone.0039898.s006" target="_blank">figure S6</a> and the densitometric analysis results are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039898#pone.0039898.s012" target="_blank">dataset S2</a>.</p

Results of siRNA mediated experimental inhibition and respective in-silico reconstruction of concentration inhibition of kinases.

<p>Effect of experimental depletion of Raf-1 (A), PI-3K (B) and p38MAPK (C) concentrations by siRNA inhibition in P388D1, a macrophage cell line is shown. The figures show the phosphorylation ratio (phosphorylated kinase/total kinase) of the kinases at uninhibited and siRNA mediated inhibition of the kinases (The representative western blots are provided as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039898#pone.0039898.s009" target="_blank">figure S9</a> and densitometric analysis results are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039898#pone.0039898.s011" target="_blank">dataset S1</a>). (D) The extent of concentration depletion for the kinases subjected to siRNA inhibition obtained through densitometric analysis. Dose response behaviour of the model for 15 minutes stimulation time when Raf-1 (E), PI3-K (F), p38MAPK (G) concentration was depleted in the same fraction as shown in (D). Here “U-In” and “si-” means un-inhibited and siRNA inhibited respectively.</p