Oncologic Trogocytosis of an Original Stromal Cells Induces Chemoresistance of Ovarian Tumours

Background

The microenvironment plays a major role in the onset and progression of metastasis. Epithelial ovarian cancer (EOC) tends to metastasize to the peritoneal cavity where interactions within the microenvironment might lead to chemoresistance. Mesothelial cells are important actors of the peritoneal homeostasis; we determined their role in the acquisition of chemoresistance of ovarian tumours.

Methodology/Principal Findings

We isolated an original type of stromal cells, referred to as “Hospicells” from ascitis of patients with ovarian carcinosis using limiting dilution. We studied their ability to confer chemoresistance through heterocellular interactions. These stromal cells displayed a new phenotype with positive immunostaining for CD9, CD10, CD29, CD146, CD166 and Multi drug resistance protein. They preferentially interacted with epithelial ovarian cancer cells. This interaction induced chemoresistance to platin and taxans with the implication of multi-drug resistance proteins. This contact enabled EOC cells to capture patches of the Hospicells membrane through oncologic trogocytosis, therefore acquiring their functional P-gp proteins and thus developing chemoresistance. Presence of Hospicells on ovarian cancer tissue micro-array from patients with neo-adjuvant chemotherapy was also significantly associated to chemoresistance.

Conclusions/Significance

This is the first report of trogocytosis occurring between a cancer cell and an original type of stromal cell. This interaction induced autonomous acquisition of chemoresistance. The presence of stromal cells within patient''s tumour might be predictive of chemoresistance. The specific interaction between cancer cells and stromal cells might be targeted during chemotherapy.     

Reflux of Endoplasmic Reticulum proteins to the cytosol inactivates tumor suppressors

In the past decades, many studies reported the presence of endoplasmic reticulum (ER)‐resident proteins in the cytosol. However, the mechanisms by which these proteins relocate and whether they exert cytosolic functions remain unknown. We find that a subset of ER luminal proteins accumulates in the cytosol of glioblastoma cells isolated from mouse and human tumors. In cultured cells, ER protein reflux to the cytosol occurs upon ER proteostasis perturbation. Using the ER luminal protein anterior gradient 2 (AGR2) as a proof of concept, we tested whether the refluxed proteins gain new functions in the cytosol. We find that refluxed, cytosolic AGR2 binds and inhibits the tumor suppressor p53. These data suggest that ER reflux constitutes an ER surveillance mechanism to relieve the ER from its contents upon stress, providing a selective advantage to tumor cells through gain‐of‐cytosolic functions—a phenomenon we name ER to Cytosol Signaling (ERCYS).     

The Effect of Anti-Synaptosomal Membrane Antibodies on Neurotransmitter Uptake

Antibodies raised against synaptosomal plasma membranes of rat hippocampus (anti-HPC IgG) caused inhibition of [3H]noradrenaline, [3H]5-hydroxytryptamine, [3H]GABA and [3H]aspartate uptake into S1 fractions and slices of hippocampus and cerebral cortex, but not those of caudate nucleus and hypothalamus. Similar inhibition was not observed on using antibodies against synaptosomal membranes of rat caudate nucleus. Anti-HPC IgG raised against synaptosomal membranes of hippocampus failed to alter both spontaneous and K+-evoked release of [3H]noradrenaline. They did not interfere with the binding of [3H]desipramine (the potent noradrenaline-uptake inhibitor) and with the binding of [3H]dihydroalprenolol, thus excluding any interaction of the antibodies with drug receptors which are located on either the pre- or postsynaptic membrane. The anti-HPC IgG inhibit the enzymatic activity of [Na+-K+-]ATPase by 30% upon incubation of the antibodies with crude membrane preparations. A comparison of their inhibitory effects with those of the neurotoxin 6-hydroxydopamine suggests that the corresponding hippocampal specific antigens are located at a presynaptic site.     

Versatile derivatives of carbohydrate-binding modules for imaging of complex carbohydrates approaching the molecular level of resolution

The innate binding specificity of different carbohydrate-binding modules (CBMs) offers a versatile approach for mapping the chemistry and structure of surfaces that contain complex carbohydrates. We have employed the distinct recognition properties of a double His-tagged recombinant CBM tagged with semiconductor quantum dots for direct imaging of crystalline cellulose at the molecular level of resolution, using transmission and scanning transmission electron microscopy. In addition, three different types of CBMs from families 3, 6, and 20 that exhibit different carbohydrate specificities were each fused with either green fluorescent protein (GFP) or red fluorescent protein (RFP) and employed for double-labeling fluorescence microscopy studies of primary cell walls and various mixtures of complex carbohydrate target molecules. CBM probes can be used for characterizing both native complex carbohydrates and engineered biomaterials.     

Structural and functional recovery of electropermeabilized skeletal muscle in-vivo after treatment with surfactant poloxamer 188

A critical requirement for cell survival after trauma is sealing of breaks in the cell membrane [M. Bier, S.M. Hammer, D.J. Canaday, R.C Lee, Kinetics of sealing for transient electropores in isolated mammalian skeletal muscle cells, Bioelectromagnetics 20 (1999) 194-201; R.C. Lee, D.C. Gaylor, D. Bhatt, D.A. Israel, Role of cell membrane rupture in the pathogenesis of electrical trauma, J. Surg. Res. 44 (1988) 709-719; R.C. Lee, J.F. Burke, E.G. Cravalho (Eds.), Electrical Trauma: The Pathophysiology, Manifestations, and Clinical Management, Cambridge University Press, 1992; B.I. Tropea, R.C. Lee, Thermal injury kinetics in electrical trauma, J. Biomech. Engr. 114 (1992) 241-250; F. Despa, D.P. Orgill, J. Newalder, R.C Lee, The relative thermal stability of tissue macromolecules and cellular structure in burn injury, Burns 31 (2005) 568-577; T.A. Block, J.N. Aarsvold, K.L. Matthews II, R.A. Mintzer, L.P. River, M. Capelli-Schellpfeffer, R.L. Wollman, S. Tripathi, C.T. Chen, R.C. Lee, The 1995 Lindberg Award. Nonthermally mediated muscle injury and necrosis in electrical trauma, J. Burn Care and Rehabil. 16 (1995) 581-588; K. Miyake, P.L. McNeil, Mechanical injury and repair of cells, Crit. Care Med. 31 (2003) S496-S501; R.C. Lee, L.P. River, F.S. Pan, R.L. Wollmann, Surfactant-induced sealing of electropermeabilized skeletal muscle membranes in vivo, Proc. Natl. Acad. Sci. 89 (1992) 4524-4528; J.D. Marks, C.Y. Pan, T. Bushell, W. Cromie, R.C. Lee, Amphiphilic, tri-block copolymers provide potent membrane-targeted neuroprotection, FASEB J. 15 (2001) 1107-1109; B. Greenebaum, K. Blossfield, J. Hannig, C.S. Carrillo, M.A. Beckett, R.R. Weichselbaum, R.C. Lee, Poloxamer 188 prevents acute necrosis of adult skeletal muscle cells following high-dose irradiation, Burns 30 (2004) 539-547; G. Serbest, J. Horwitz, K. Barbee, The effect of poloxamer-188 on neuronal cell recovery from mechanical injury, J. Neurotrauma 22 (2005) 119-132]. The triblock copolymer surfactant Poloxamer 188 (P188) is known to increase the cell survival after membrane electroporation [R.C. Lee, L.P. River, F.S. Pan, R.L. Wollmann, Surfactant-induced sealing of electropermeabilized skeletal muscle membranes in vivo, Proc. Natl. Acad. Sci. 89 (1992) 4524-4528; Z. Ababneh, H. Beloeil, C.B. Berde, G. Gambarota, S.E. Maier, R.V. Mulkern, Biexponential parametrization of T2 and diffusion decay curves in a rat muscle edema model: Decay curve components and water compartments, Magn. Reson. Med. 54 (2005) 524-531]. Here, we use a rat hind-limb model of electroporation injury to determine if the intravenous administration of P188 improves the recovery of the muscle function. Rat hind-limbs received a sequence of either 0, 3, 6, 9, or 12 electrical current pulses (2 A, 4 ms duration, 10 s duty cycle). Magnetic resonance imaging (MRI) analysis, muscle water content and compound muscle action potential (CMAP) amplitudes were compared. Electroporation injury manifested edema formation and depression of the CMAP amplitudes. P188 (one bolus of 1 mg/ml of blood) was administrated 30 or 60 min after injury. Animals receiving P188 exhibited reduced tissue edema (p<0.05) and increased CMAP amplitudes (p<0.03). By comparison, treatment with 10 kDa neutral dextran, which produces similar serum osmotic effects as P188, had no effect on post-electroporation recovery. Noteworthy, the present results suggest that a single intravenous dose of P188 is effective to restore the structural integrity of damaged tissues with intact circulation.     

Quantitative Phosphoproteomics Reveals SLP-76 Dependent Regulation of PAG and Src Family Kinases in T Cells

The SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) plays a critical scaffolding role in T cell receptor (TCR) signaling. As an adaptor protein that contains multiple protein-binding domains, SLP-76 interacts with many signaling molecules and links proximal receptor stimulation to downstream effectors. The function of SLP-76 in TCR signaling has been widely studied using the Jurkat human leukaemic T cell line through protein disruption or site-directed mutagenesis. However, a wide-scale characterization of SLP-76-dependant phosphorylation events is still lacking. Quantitative profiling of over a hundred tyrosine phosphorylation sites revealed new modes of regulation of phosphorylation of PAG, PI3K, and WASP while reconfirming previously established regulation of Itk, PLCγ, and Erk phosphorylation by SLP-76. The absence of SLP-76 also perturbed the phosphorylation of Src family kinases (SFKs) Lck and Fyn, and subsequently a large number of SFK-regulated signaling molecules. Altogether our data suggests unique modes of regulation of positive and negative feedback pathways in T cells by SLP-76, reconfirming its central role in the pathway.     

Development of an improved selective agar medium for isolation of Yersinia pestis

Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.     

Protein phosphatase 2A (PP2A)-specific ubiquitin ligase MID1 is a sequence-dependent regulator of translation efficiency controlling 3-phosphoinositide-dependent protein kinase-1 (PDPK-1)

We have shown previously that the ubiquitin ligase MID1, mutations of which cause the midline malformation Opitz BBB/G syndrome (OS), serves as scaffold for a microtubule-associated protein complex that regulates protein phosphatase 2A (PP2A) activity in a ubiquitin-dependent manner. Here, we show that the MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) and thereby increases stability and translational efficiency of these mRNAs. Strikingly, inclusion of multiple copies of the MIDAS motif into mammalian mRNAs increases production of the encoded proteins up to 20-fold. Mutated MID1, as found in OS patients, loses its influence on MIDAS-containing mRNAs, suggesting that the malformations in OS patients could be caused by failures in the regulation of cytoskeleton-bound protein translation. This is supported by the observation that the majority of mRNAs that carry MIDAS motifs is involved in developmental processes and/or energy homeostasis. Further analysis of one of the proteins encoded by a MIDAS-containing mRNA, namely PDPK-1 (3-phosphoinositide dependent protein kinase-1), which is an important regulator of mammalian target of rapamycin/PP2A signaling, showed that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient compared with an age-matched control and can be rescued by functional MID1. Together, our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation. They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells.