Adaptive resistance of melanoma cells to RAF inhibition via reversible induction of a slowly dividing de-differentiated state.

Treatment of BRAF-mutant melanomas with MAP kinase pathway inhibitors is paradigmatic of the promise of precision cancer therapy but also highlights problems with drug resistance that limit patient benefit. We use live-cell imaging, single-cell analysis, and molecular profiling to show that exposure of tumor cells to RAF/MEK inhibitors elicits a heterogeneous response in which some cells die, some arrest, and the remainder adapt to drug. Drug-adapted cells up-regulate markers of the neural crest (e.g., NGFR), a melanocyte precursor, and grow slowly. This phenotype is transiently stable, reverting to the drug-naïve state within 9 days of drug withdrawal. Transcriptional profiling of cell lines and human tumors implicates a c-Jun/ECM/FAK/Src cascade in de-differentiation in about one-third of cell lines studied; drug-induced changes in c-Jun and NGFR levels are also observed in xenograft and human tumors. Drugs targeting the c-Jun/ECM/FAK/Src cascade as well as BET bromodomain inhibitors increase the maximum effect (Emax) of RAF/MEK kinase inhibitors by promoting cell killing. Thus, analysis of reversible drug resistance at a single-cell level identifies signaling pathways and inhibitory drugs missed by assays that focus on cell populations.

An unusual form of reaction wood in Koromiko [Hebe salicifolia G. Forst. (Pennell)], a southern hemisphere angiosperm.

Koromiko [Hebe salicifolia G. Forst. (Pennell)] is a woody angiosperm native to New Zealand and Chile. Hebe spp. belong to the otherwise herbaceous family Plantaginaceae in the order Lamiales. Reaction wood exerting expansional forces was found on the lower side of leaning H. salicifolia stems. Such reaction wood is atypical for angiosperms, which commonly form contracting reaction wood on the upper side of leaning stems. Reaction wood typical for angiosperms is formed by species in other families in the order Lamiales. This suggests that the form of reaction wood is specific to the family level. Functionally the reaction wood of H. salicifolia is similar to that found in gymnosperms, which both act by pushing. However, their chemical, anatomical and physical characteristics are different. Typical features of reaction wood present in gymnosperms such as high density, thick-walled rounded cells and the presence of (1 → 4)-ß-galactan in the secondary cell wall layer are absent in H. salicifolia reaction wood. Reaction wood of H. salicifolia varies from normal wood in having a higher microfibril angle, which is likely to determine the direction of generated maturation stresses.

Receptor dynamics in signaling.

Reliable inter- and intracellular communication is central to both the development and the integrity of multicellular organisms. Key mediators of these processes are cell surface receptors that perceive and convert extracellular cues to trigger intracellular signaling networks and ultimately a phenotypic response. Deregulation of signal transduction leads to a variety of diseases, and aberrations in receptor proteins are very common in various cancer types. Therefore, cell surface receptors have been established as major targets in drug discovery. However, in order to efficiently apply therapeutics, it is crucial to gain knowledge about design principles of receptor signaling. In this chapter, we will discuss signal transduction at the receptor level for examples from different receptor classes.

Stat5 activation enables erythropoiesis in the absence of EpoR and Jak2.

Erythropoiesis requires erythropoietin (Epo) and stem cell factor (SCF) signaling via their receptors EpoR and c-Kit. EpoR, like many other receptors involved in hematopoiesis, acts via the kinase Jak2. Deletion of EpoR or Janus kinase 2 (Jak2) causes embryonic lethality as a result of defective erythropoiesis. The contribution of distinct EpoR/Jak2-induced signaling pathways (mitogen-activated protein kinase, phosphatidylinositol 3-kinase, signal transducer and activator of transcription 5 [Stat5]) to functional erythropoiesis is incompletely understood. Here we demonstrate that expression of a constitutively activated Stat5a mutant (cS5) was sufficient to relieve the proliferation defect of Jak2(-/-) and EpoR(-/-) cells in an Epo-independent manner. In addition, tamoxifen-induced DNA binding of a Stat5a-estrogen receptor (ER)* fusion construct enabled erythropoiesis in the absence of Epo. Furthermore, c-Kit was able to enhance signaling through the Jak2-Stat5 axis, particularly in lymphoid and myeloid progenitors. Although abundance of hematopoietic stem cells was 2.5-fold reduced in Jak2(-/-) fetal livers, transplantation of Jak2(-/-)-cS5 fetal liver cells into irradiated mice gave rise to mature erythroid and myeloid cells of donor origin up to 6 months after transplantation. Cytokine- and c-Kit pathways do not function independently of each other in hematopoiesis but cooperate to attain full Jak2/Stat5 activation. In conclusion, activated Stat5 is a critical downstream effector of Jak2 in erythropoiesis/myelopoiesis, and Jak2 functionally links cytokine- with c-Kit-receptor tyrosine kinase signaling.

Packing density of the erythropoietin receptor transmembrane domain correlates with amplification of biological responses.

The formation of signal-promoting dimeric or oligomeric receptor complexes at the cell surface is modulated by self-interaction of their transmembrane (TM) domains. To address the importance of TM domain packing density for receptor functionality, we examined a set of asparagine mutants in the TM domain of the erythropoietin receptor (EpoR). We identified EpoR-T242N as a receptor variant that is present at the cell surface similar to wild-type EpoR but lacks visible localization in vesicle-like structures and is impaired in efficient activation of specific signaling cascades. Analysis by a molecular modeling approach indicated an increased interhelical distance for the EpoR-T242N TM dimer. By employing the model, we designed additional mutants with increased or decreased packing volume and confirmed a correlation between packing volume and biological responsiveness. These results propose that the packing density of the TM domain provides a novel layer for fine-tuned regulation of signal transduction and cellular decisions.

Combined effects of soil salinity and high temperature on photosynthesis and growth of quinoa plants (Chenopodium quinoa).

The halophytic crop quinoa (Chenopodium quinoa Willd.) is adapted to soil salinity and cold climate, but recent investigations have shown that quinoa can be grown in significantly warmer latitudes, i.e. the Mediterranean region, where high temperature and soil salinity can occur in combination. In this greenhouse study, effects of saltwater irrigation and high temperature on growth and development of the Bolivian cultivar 'Achachino' were determined. Development was slightly delayed in response to saltwater treatment, but significantly faster at high temperature. Biomass and seed yield decreased in response to salt, but not to high temperature. Plants increased their number of stomata in response to salt stress, but reduced its size on both sides of the leaf, whereas high temperature treatment significantly increased the stomata size on the abaxial leaf surface. When salt and high temperature was combined, the size of stomata was reduced only on the abaxial side of the leaf, and the number of epidermal bladder cells significantly increased on the abaxial leaf surface, resulting in preservation of photosynthetic quantum yields. We hypothesise that this morphological plasticity improves the partition of water and CO2 resulting in maintenance of photosynthesis in quinoa under adverse environmental conditions. We present a GLM-model that predicts yield parameters of quinoa grown in regions affected by soil salinity, high temperature and the factors combined.

Multiplexed Exchange-PAINT imaging reveals ligand-dependent EGFR and Met interactions in the plasma membrane.

Signal transduction by receptor tyrosine kinases (RTKs) involves complex ligand- and time-dependent changes in conformation and modification state. High resolution structures are available for individual receptors dimers, but less is known about receptor clusters that form in plasma membranes composed of many different RTKs with the potential to interact. We report the use of multiplexed super-resolution imaging (Exchange-PAINT) followed by mean-shift clustering and random forest analysis to measure the precise distributions of five receptor tyrosine kinases (RTKs) from the ErbB, IGF-1R and Met families in breast cancer cells. We find that these receptors are intermixed nonhomogenously on the plasma membrane. Stimulation by EGF does not appear to induce a change in the density of EGFR in local clusters but instead results in formation of EGFR-Met and EGFR-ErbB3 associations; non-canonical EGFR-Met interactions are implicated in resistance to anti-cancer drugs but have not been previously detected in drug-naïve cells.

Electrical signaling along the phloem and its physiological responses in the maize leaf.

To elucidate the role of electrical signaling in the phloem of maize the tips of attached leaves were stimulated by chilling and wounding. Two different signals were detected in the phloem at the middle of the leaf using the aphid stylet technique: (1) action potentials (AP) arose in the phloem after chilling; and (2) variation potentials (VPs) were evoked after wounding the leaf tip. Combined electric potential and gas exchange measurements showed that while the wound-induced VP moved rapidly towards the middle of the leaf to induce a reduction in both the net-CO2 uptake rate and the stomatal conductance, there was no response in the gas exchange to the cold-induced AP. To determine if electrical signaling had any impact on assimilate transport the middle of the leaf was exposed to (14)CO2. Autoradiography of labeled assimilates provided evidence that phloem and intercellular transport of assimilates from mesophyll to bundle sheath cells was strongly reduced while the cold-induced AP moved through. In contrast, wound-induced VP did not inhibit assimilate translocation but did reduce the amount of the labeled assimilate in phloem and bundle sheath cells. Biochemical analysis revealed that callose content increased significantly in chilled leaves while starch increased in chilled but decreased in wounded leaves. The results led to the conclusion that different stimulation types incite characteristic phloem-transmitted electrical signals, each with a specific influence on gas exchange and assimilate transport.

Feasibility of fecal microRNAs as novel biomarkers for pancreatic cancer.

INTRODUCTION: Pancreatic cancer (PCA) is an aggressive tumor that associates with high mortality rates. Majority of PCA patients are diagnosed usually at late tumor stages when the therapeutic options are limited. MicroRNAs (miRNA) are involved in tumor development and are commonly dysregulated in PCA. As a proof-of-principle study, we aimed to evaluate the potential of fecal miRNAs as biomarkers for pancreatic cancer. MATERIALS AND METHODS: Total RNA was extracted from feces using Qiagen's miRNA Mini Kit. For miRNA expression analyses we selected a subset of 7 miRNAs that are frequently dysregulated in PCA (miR-21, -143, -155, -196a, -210, -216a, -375). Subsequently, expression levels of these miRNAs were determined in fecal samples from controls (n = 15), chronic pancreatitis (n = 15) and PCA patients (n = 15) using quantitative TaqMan-PCR assays. RESULTS: All selected miRNAs were detectable in fecal samples with high reproducibility. Four of seven miRNAs (miR-216a, -196a, -143 und -155) were detected at lower concentrations in feces of PCA patients when compared to controls (p<0.05). Analysis of fecal miRNA expression in controls and patients with chronic pancreatitis and PCA revealed that the expression of miR-216a, -196a, -143 und -155 were highest in controls and lowest in PCA. The expression of the remaining three miRNAs (miR-21, -210 and -375) remained unchanged among controls and the patients with either chronic pancreatitis or PCA. CONCLUSION: Our data provide novel evidence for the differential expression of miRNAs in feces of patients with PCA. If successfully validated in large-scale prospective studies, the fecal miRNA biomarkers may offer novel tools for PCA screening research.

In silico labeling reveals the time-dependent label half-life and transit-time in dynamical systems.

BACKGROUND: Mathematical models of dynamical systems facilitate the computation of characteristic properties that are not accessible experimentally. In cell biology, two main properties of interest are (1) the time-period a protein is accessible to other molecules in a certain state - its half-life - and (2) the time it spends when passing through a subsystem - its transit-time. We discuss two approaches to quantify the half-life, present the novel method of in silico labeling, and introduce the label half-life and label transit-time. The developed method has been motivated by laboratory tracer experiments. To investigate the kinetic properties and behavior of a substance of interest, we computationally label this species in order to track it throughout its life cycle. The corresponding mathematical model is extended by an additional set of reactions for the labeled species, avoiding any double-counting within closed circuits, correcting for the influences of upstream fluxes, and taking into account combinatorial multiplicity for complexes or reactions with several reactants or products. A profile likelihood approach is used to estimate confidence intervals on the label half-life and transit-time. RESULTS: Application to the JAK-STAT signaling pathway in Epo-stimulated BaF3-EpoR cells enabled the calculation of the time-dependent label half-life and transit-time of STAT species. The results were robust against parameter uncertainties. CONCLUSIONS: Our approach renders possible the estimation of species and label half-lives and transit-times. It is applicable to large non-linear systems and an implementation is provided within the PottersWheel modeling framework (http://www.potterswheel.de).

Prognostic significance of erythropoietin in pancreatic adenocarcinoma.

BACKGROUND: Erythropoietin (Epo) administration has been reported to have tumor-promoting effects in anemic cancer patients. We investigated the prognostic impact of endogenous Epo in patients with pancreatic ductal adenocarcinoma (PDAC). METHODOLOGY: The clinico-pathological relevance of hemoglobin (Hb, n = 150), serum Epo (sEpo, n = 87) and tissue expression of Epo/Epo receptor (EpoR, n = 104) was analyzed in patients with PDAC. Epo/EpoR expression, signaling, growth, invasion and chemoresistance were studied in Epo-exposed PDAC cell lines. RESULTS: Compared to donors, median preoperative Hb levels were reduced by 15% in both chronic pancreatitis (CP, p<0.05) and PDAC (p<0.001), reaching anemic grade in one third of patients. While inversely correlating to Hb (r = -0.46), 95% of sEPO values lay within the normal range. The individual levels of compensation were adequate in CP (observed to predicted ratio, O/P = 0.99) but not in PDAC (O/P = 0.85). Strikingly, lower sEPO values yielding inadequate Epo responses were prominent in non-metastatic M0-patients, whereas these parameters were restored in metastatic M1-group (8 vs. 13 mU/mL; O/P = 0.82 vs. 0.96; p<0.01)--although Hb levels and the prevalence of anemia were comparable. Higher sEpo values (upper quartile ≥ 16 mU/ml) were not significantly different in M0 (20%) and M1 (30%) groups, but were an independent prognostic factor for shorter survival (HR 2.20, 10 vs. 17 months, p<0.05). The pattern of Epo expression in pancreas and liver suggested ectopic release of Epo by capillaries/vasa vasorum and hepatocytes, regulated by but not emanating from tumor cells. Epo could initiate PI3K/Akt signaling via EpoR in PDAC cells but failed to alter their functions, probably due to co-expression of the soluble EpoR isoform, known to antagonize Epo. CONCLUSION/SIGNIFICANCE: Higher sEPO levels counteract anemia but worsen outcome in PDAC patients. Further trials are required to clarify how overcoming a sEPO threshold ≥16 mU/ml by endogenous or exogenous means may predispose to or promote metastatic progression.

Covering a broad dynamic range: information processing at the erythropoietin receptor.

Cell surface receptors convert extracellular cues into receptor activation, thereby triggering intracellular signaling networks and controlling cellular decisions. A major unresolved issue is the identification of receptor properties that critically determine processing of ligand-encoded information. We show by mathematical modeling of quantitative data and experimental validation that rapid ligand depletion and replenishment of the cell surface receptor are characteristic features of the erythropoietin (Epo) receptor (EpoR). The amount of Epo-EpoR complexes and EpoR activation integrated over time corresponds linearly to ligand input; this process is carried out over a broad range of ligand concentrations. This relation depends solely on EpoR turnover independent of ligand binding, which suggests an essential role of large intracellular receptor pools. These receptor properties enable the system to cope with basal and acute demand in the hematopoietic system.

A systems biology approach to analyse amplification in the JAK2-STAT5 signalling pathway.

BACKGROUND: The amplification of signals, defined as an increase in the intensity of a signal through networks of intracellular reactions, is considered one of the essential properties in many cell signalling pathways. Despite of the apparent importance of signal amplification, there have been few attempts to formalise this concept. RESULTS: In this work we investigate the amplification and responsiveness of the JAK2-STAT5 pathway using a kinetic model. The recruitment of EpoR to the plasma membrane, activation by Epo, and deactivation of the EpoR/JAK2 complex are considered as well as the activation and nucleocytoplasmic shuttling of STAT5. Using qualitative biological knowledge, we first establish the structure of a general power-law model. We then generate a family of models from which we select suitable candidates. The parameter values of the model are estimated from experimental quantitative time-course data. The final model, whether it is conventional model with fixed predefined integer kinetic orders or a model with variable non-integer kinetic orders, is selected on the basis of a good agreement between simulations and the experimental data. The model is used to analyse the responsiveness and amplification properties of the pathway with sustained, transient, and oscillatory stimulation. CONCLUSION: The selected kinetic model predicts that the system acts as an amplifier with maximum amplification and sensitivity for input signals whose intensity match physiological values for Epo concentration and with duration in the range of one to 100 minutes. The response of the system reaches saturation for more intense and longer stimulation with Epo. We hypothesise that these properties of the system directly relate to the saturation of Epo receptor activation, its low recruitment to the plasma membrane and intense deactivation as predicted by the model.

The interface between self-assembling erythropoietin receptor transmembrane segments corresponds to a membrane-spanning leucine zipper.

Structural and functional studies recently indicated that the erythropoietin receptor exists as a preassembled homodimer whose activation by ligand binding requires self-interaction of its transmembrane segment. Here, we probed the interface formed by the transmembrane segments by asparagine-scanning mutagenesis in a natural membrane. We show that this interface is based on a leucine zipper-like heptad repeat pattern of amino acids. The strongest impact of asparagine was observed at position 241, suggesting the highest packing density around this position, which is in agreement with results obtained upon mutation to alanine. Interestingly, the same face of the transmembrane helix had previously been shown to enter a heterophilic interaction with the transmembrane segment of gp55-P, a viral membrane protein that leads to ligand-independent receptor activation in infected cells. Further, functional characterization of an erythropoietin receptor mutant with asparagine at position 241 in a hematopoietic cell line showed that this protein could still be activated by erythropoietin yet was not constitutively active. This suggests that forced self-interaction of the transmembrane segments does not suffice to induce signaling of the erythropoietin receptor.

A functional green fluorescent protein-erythropoietin receptor despite physical separation of JAK2 binding site and tyrosine residues.

Signaling through hematopoietic cytokine receptors such as the erythropoietin receptor (EpoR) depends on the activation of a receptor-bound Janus kinase (JAK) and tyrosine phosphorylation of the cytoplasmic domain. To visualize the EpoR and elucidate structural requirements coordinating signal transduction, we probed the EpoR by inserting the green fluorescent protein (GFP) at various positions. We show that insertion of GFP in proximity to the transmembrane domain, either in the extracellular or the cytoplasmic domain, results in EpoR-GFP receptors incompetent to elicit biological responses in a factor-dependent cell line or in erythroid progenitor cells. Surprisingly, a receptor harboring GFP insertion in the middle of the cytoplasmic domain, and thereby separating the JAK2 binding site from the tyrosine residues, is capable of supporting signal transduction in response to ligand binding. Comparable with the wild type EpoR, but more efficient than a C-terminal EpoR-GFP fusion, this chimeric receptor promotes the maturation of erythroid progenitor cells and is localized in punctated endosome-like structures. We conclude that the extracellular, transmembrane, and membrane-proximal segment of the cytoplasmic domain form a rigid structural entity whose precise orientation is essential for the initiation of signal transduction, whereas the cytoplasmic domain possesses flexibility in adopting an activated conformation.