Expanding the Interactome of TES by Exploiting TES Modules with Different Subcellular Localizations.

The multimodular nature of many eukaryotic proteins underlies their temporal or spatial engagement in a range of protein cocomplexes. Using the multimodule protein testin (TES), we here report a proteomics approach to increase insight in cocomplex diversity. The LIM-domain containing and tumor suppressor protein TES is present at different actin cytoskeleton adhesion structures in cells and influences cell migration, adhesion and spreading. TES module accessibility has been proposed to vary due to conformational switching and variants of TES lacking specific domains target to different subcellular locations. By applying iMixPro AP-MS ("intelligent Mixing of Proteomes"-affinity purification-mass spectrometry) to a set of tagged-TES modular variants, we identified proteins residing in module-specific cocomplexes. The obtained distinct module-specific interactomes combine to a global TES interactome that becomes more extensive and richer in information. Applying pathway analysis to the module interactomes revealed expected actin-related canonical pathways and also less expected pathways. We validated two new TES cocomplex partners: TGFB1I1 and a short form of the glucocorticoid receptor. TES and TGFB1I1 are shown to oppositely affect cell spreading providing biological validity for their copresence in complexes since they act in similar processes.

A high rate of telomeric sister chromatid exchange occurs in chronic lymphocytic leukaemia B-cells.

Cancer cells protect their telomere ends from erosion through reactivation of telomerase or by using the Alternative Lengthening of Telomere (ALT) mechanism that depends on homologous recombination. Chronic lymphocytic leukaemia (CLL) B cells are characterized by almost no telomerase activity, shelterin deregulation and telomere fusions. To characterize telomeric maintenance mechanisms in B-CLL patients, we measured their telomere length, telomerase expression and the main hallmarks of the ALT activity i.e. C-circle concentration, an extra-chromosomal telomere repeat (ECTR), and the level of telomeric sister chromatid exchange (T-SCE) rate. Patients showed relative homogenous telomere length although almost no TERT transcript and nearly no C-circle were evidenced. Nevertheless, compared with normal B cells, B-CLL cells showed an increase in T-SCE rate that was correlated with a strong down-regulation of the topoisomerase III alpha (TOP3A) expression, involved in the dissolution of Holliday Junctions (HJ), together with an increased expression of SLX1A, SLX4, MUS81 and GEN1, involved in the resolution of HJ. Altogether, our results suggest that the telomere maintenance mechanism of B-CLL cells do not preferentially use telomerase or ALT. Rather, the rupture of the dissolvasome/resolvasome balance may increase telomere shuffling that could homogenize telomere length, slowing telomere erosion in this disease.

Granzyme B degradation by autophagy decreases tumor cell susceptibility to natural killer-mediated lysis under hypoxia.

Recent studies demonstrated that autophagy is an important regulator of innate immune response. However, the mechanism by which autophagy regulates natural killer (NK) cell-mediated antitumor immune responses remains elusive. Here, we demonstrate that hypoxia impairs breast cancer cell susceptibility to NK-mediated lysis in vitro via the activation of autophagy. This impairment was not related to a defect in target cell recognition by NK cells but to the degradation of NK-derived granzyme B in autophagosomes of hypoxic cells. Inhibition of autophagy by targeting beclin1 (BECN1) restored granzyme B levels in hypoxic cells in vitro and induced tumor regression in vivo by facilitating NK-mediated tumor cell killing. Together, our data highlight autophagy as a mechanism underlying the resistance of hypoxic tumor cells to NK-mediated lysis. The work presented here provides a cutting-edge advance in our understanding of the mechanism by which hypoxia-induced autophagy impairs NK-mediated lysis in vitro and paves the way for the formulation of more effective NK cell-based antitumor therapies.

Tyrosine kinase gene fusions in cancer: translating mechanisms into targeted therapies.

Tyrosine kinase fusion genes represent an important class of oncogenes associated with leukaemia and solid tumours. They are produced by translocations and other chromosomal rearrangements of a subset of tyrosine kinase genes, including ABL, PDGFRA, PDGFRB, FGFR1, SYK, RET, JAK2 and ALK. Based on recent findings, this review discusses the common mechanisms of activation of these fusion genes. Enforced oligomerization and inactivation of inhibitory domains are the two key processes that switch on the kinase domain. Activated tyrosine kinase fusions then signal via an array of transduction cascades, which are largely shared. In addition, the fusion partner provides a scaffold for the recruitment of proteins that contribute to signalling, protein stability, cellular localization and oligomerization. The expression level of the fusion protein is another critical parameter. Its transcription is controlled by the partner gene promoter, while translation may be regulated by miRNA. Several mechanisms also prevent the degradation of the oncoprotein by proteasomes and lysosomes, leading to its accumulation in cells. The selective inhibition of the tyrosine kinase activity by adenosine-5'-triphosphate competitors, such as imatinib, is a major therapeutic success. Imatinib induces remission in leukaemia patients that are positive for BCR-ABL or PDGFR fusions. Recently, crizotinib produced promising results in a subtype of lung cancers with ALK fusion. However, resistance was reported in both cases, partially due to mutations. To tackle this problem, additional levels of therapeutic interventions are suggested by the complex mechanisms of fusion tyrosine kinase activation. New approaches include allosteric inhibition and interfering with oligomerization or chaperones.

Multiple oligomerization domains of KANK1-PDGFRß are required for JAK2-independent hematopoietic cell proliferation and signaling via STAT5 and ERK.

BACKGROUND: KANK1-PDGFRB is a fusion gene generated by the t(5;9) translocation between KANK1 and the platelet-derived growth factor receptor beta gene PDGFRB. This hybrid was identified in a myeloproliferative neoplasm featuring severe thrombocythemia, in the absence of the JAK2 V617F mutation. DESIGN AND METHODS: KANK1-PDGFRB was transduced into Ba/F3 cells and CD34(+) human progenitor cells to gain insights into the mechanisms whereby this fusion gene transforms cells. RESULTS: Although platelet-derived growth factor receptors are capable of activating JAK2, KANK1-PDGFRß did not induce JAK2 phosphorylation in hematopoietic cells and a JAK inhibitor did not affect KANK1-PDGFRß-induced cell growth. Like JAK2 V617F, KANK1-PDGFRß constitutively activated STAT5 transcription factors, but this did not require JAK kinases. In addition KANK1-PDGFRß induced the phosphorylation of phospholipase C-γ, ERK1 and ERK2, like wild-type PDGFRß and TEL-PDGFRß, another hybrid protein found in myeloid malignancies. We next tested various mutant forms of KANK1-PDGFRß in Ba/F3 cells and human CD34(+) hematopoietic progenitors. The three coiled-coil domains located in the N-terminus of KANK1 were required for KANK1-PDGFRß-induced cell growth and signaling via STAT5 and ERK. However, the coiled-coils were not essential for KANK1-PDGFRß oligomerization, which could be mediated by another new oligomerization domain. KANK1-PDGFRß formed homotrimeric complexes and heavier oligomers. CONCLUSIONS: KANK1-PDGFRB is a unique example of a thrombocythemia-associated oncogene that does not signal via JAK2. The fusion protein is activated by multiple oligomerization domains, which are required for signaling and cell growth stimulation.

[Role of PDGF and FGF receptors in cancer]. / Role des récepteurs du PDGF et du FGF dans les cancers.

Growth factors of the PDGF and FGF families act through receptor tyrosine kinases. These receptors can be activated by chromosomal rearrangements in myeloid neoplasms associated with hypereosinophilia. We identified a new fusion gene between KANK1 and PDGFRbeta in a patient with thrombocythemia. We showed that such fusion oncoproteins derived from PDGF and FGF receptors escape the normal degradation pathways, leading to their accumulation in cells. This process amplifies signalling leading to cell proliferation. Using microarrays and bioinformatics, we showed that several transcription factors contribute to the control cell growth, including STATS, FOXO and SREBP.

Rapid multiplex gene expression assays for monitoring metabolic resistance in the major malaria vector Anopheles gambiae.

BACKGROUND: Metabolic resistance of the major malaria vector Anopheles gambiae (s.l.) to insecticides is operationally significant, particularly in combination with target site resistance. However, detection of metabolic resistance is not trivial and relies on laborious bioassays, unspecific biochemical methods, or sophisticated and expensive molecular approaches using transcriptomics. METHODS: Rapid one-step multiplex TaqMan-probe based RT-qPCR assays were developed and optimised to measure the expression levels of genes associated with metabolic insecticide resistance in An. gambiae (s.l.). Primers and probes were designed to target the mRNA of cytochrome P450-dependent monooxygenases CYP6P3, CYP6M2, CYP9K1, CYP6P4 and CYP6Z1, and the glutathione-S-transferase GSTE2. The novel assays were validated versus gold standard methods with a range of phenotyped mosquito specimens. The assays were also tested directly on lysates of RNAlater®-preserved mosquitoes without an RNA extraction step. RESULTS: The novel assays are efficient (reaction efficiencies = 95-109%), sensitive (covering a > 10.0 Ct range with R2 values > 0.99), specific (TaqMan chemistry), reproducible (%CV = 4.46-12.07%), as well as readily expandable to capture additional loci as they evolve or to cover additional species. The assays were successfully validated in terms of expression levels against standard two-step singleplex qPCR assays (overall % difference = -17.6%, 95% CI = -38.7-3.43%) and microarrays, using laboratory strains and field-caught samples. The assays can also be applied directly on lysates of mosquito specimens, without RNA extraction or DNase treatment. CONCLUSIONS: The novel multiplex assays for monitoring the levels of major detoxification genes and metabolic resistance in An. gambiae (s.l.) are simple to perform, robust and rapid. They may complement current diagnostic assays to provide evidence-based and operationally relevant information for insecticide resistance management.

Delineating the Tes Interaction Site in Zyxin and Studying Cellular Effects of Its Disruption.

Focal adhesions are integrin-based structures that link the actin cytoskeleton and the extracellular matrix. They play an important role in various cellular functions such as cell signaling, cell motility and cell shape. To ensure and fine tune these different cellular functions, adhesions are regulated by a large number of proteins. The LIM domain protein zyxin localizes to focal adhesions where it participates in the regulation of the actin cytoskeleton. Because of its interactions with a variety of binding partners, zyxin has been proposed to act as a molecular scaffold. Here, we studied the interaction of zyxin with such a partner: Tes. Similar to zyxin, Tes harbors three highly conserved LIM domains of which the LIM1 domain directly interacts with zyxin. Using different zyxin variants in pull-down assays and ectopic recruitment experiments, we identified the Tes binding site in zyxin and showed that four highly conserved amino acids are crucial for its interaction with Tes. Based upon these findings, we used a zyxin mutant defective in Tes-binding to assess the functional consequences of abrogating the zyxin-Tes interaction in focal adhesions. Performing fluorescence recovery after photobleaching, we showed that zyxin recruits Tes to focal adhesions and modulates its turnover in these structures. However, we also provide evidence for zyxin-independent localization of Tes to focal adhesions. Zyxin increases focal adhesion numbers and reduces focal adhesion lifetimes, but does so independent of Tes. Quantitative analysis showed that the loss of interaction between zyxin and Tes affects the process of cell spreading. We conclude that zyxin influences focal adhesion dynamics, that it recruits Tes and that this interaction is functional in regulating cell spreading.

Autophagic degradation of GZMB/granzyme B: a new mechanism of hypoxic tumor cell escape from natural killer cell-mediated lysis.

The crucial issue for defining successful natural killer (NK)-based anticancer therapy is the ability of tumor cells to activate resistance mechanisms leading to escape from NK-mediated killing. It is now well established that such mechanisms are likely evolved under hypoxia in the tumor microenvironment. Here, we show that hypoxia-induced autophagy impairs breast cancer cell susceptibility to NK-mediated lysis and that this impairment is reverted by targeting autophagy. We provide evidence that activation of autophagy in hypoxic cells is involved in selective degradation of the pro-apoptotic NK-derived serine protease GZMB/granzyme B, thereby blocking NK-mediated target cell apoptosis. Our in vivo data validate the concept that targeting autophagy in cancer cells promotes tumor regression by facilitating their elimination by NK cells. This study provides a cutting-edge advance in our understanding of how hypoxia-induced autophagy impairs NK-mediated lysis and might pave the way for formulating more effective NK-based antitumor therapy by combining autophagy inhibitors.

The critical role of the tumor microenvironment in shaping natural killer cell-mediated anti-tumor immunity.

Considerable evidence has been gathered over the last 10 years showing that the tumor microenvironment (TME) is not simply a passive recipient of immune cells, but an active participant in the establishment of immunosuppressive conditions. It is now well documented that hypoxia, within the TME, affects the functions of immune effectors including natural killer (NK) cells by multiple overlapping mechanisms. Indeed, each cell in the TME, irrespective of its transformation status, has the capacity to adapt to the hostile TME and produce immune modulatory signals or mediators affecting the function of immune cells either directly or through the stimulation of other cells present in the tumor site. This observation has led to intense research efforts focused mainly on tumor-derived factors. Notably, it has become increasingly clear that tumor cells secrete a number of environmental factors such as cytokines, growth factors, exosomes, and microRNAs impacting the immune cell response. Moreover, tumor cells in hostile microenvironments may activate their own intrinsic resistance mechanisms, such as autophagy, to escape the effective immune response. Such adaptive mechanisms may also include the ability of tumor cells to modify their metabolism and release several metabolites to impair the function of immune cells. In this review, we summarize the different mechanisms involved in the TME that affect the anti-tumor immune function of NK cells.

An integrative simulation model linking major biochemical reactions of actin-polymerization to structural properties of actin filaments.

We report on an advanced universal Monte Carlo simulation model of actin polymerization processes offering a broad application panel. The model integrates major actin-related reactions, such as assembly of actin nuclei, association/dissociation of monomers to filament ends, ATP-hydrolysis via ADP-Pi formation and ADP-ATP exchange, filament branching, fragmentation and annealing or the effects of regulatory proteins. Importantly, these reactions are linked to information on the nucleotide state of actin subunits in filaments (ATP hydrolysis) and the distribution of actin filament lengths. The developed stochastic simulation modelling schemes were validated on: i) synthetic theoretical data generated by a deterministic model and ii) sets of our and published experimental data obtained from fluorescence pyrene-actin experiments. Build on an open-architecture principle, the designed model can be extended for predictive evaluation of the activities of other actin-interacting proteins and can be applied for the analysis of experimental pyrene actin-based or fluorescence microscopy data. We provide a user-friendly, free software package ActinSimChem that integrates the implemented simulation algorithms and that is made available to the scientific community for modelling in silico any specific actin-polymerization system.