Organizing and modulatory roles of plasma membrane microdomains in cell signaling and tumor therapy

ABSZTRAKT: Az irodalomban számos publikáció mutat rá arra, hogy a sejtmembrán monosialo-tetrahexozil-gangliozidban (GM1) gazdag területei – melyek azonosíthatók az úgynevezett lipid raftokkal, vagy lipid-tutajokkal – fontos szervező-moduláló szerepet játszanak a különböző kimenetelű jelátviteli útvonalak szabályozásában. A citoplazmamembrán ezen koleszterinben és szfingolipidekben gazdag, szubmikron méretű strukturális egységeinek tehát nem pusztán morfológiai, hanem funkcionális szerepe van. A raftok képesek egymástól szigetszerűen elkülönítve akkumulálni a különböző sejtélettani folyamatokban együttműködő membránfehérjéket, így biztosítva az eltérő kimenetelű jelátviteli kaszkádok hatékony lejátszódását. Ezek a lipid domének, mint membránon úszó „forró területek” egy kis átmérőjű – 50-150 nm – területre képesek koncentrálni a receptorokat és más járulékos fehérjéket, ami egy sokkal hatékonyabb és intenzívebb lefolyású jelátvitelhez vezet. Dolgozatomban szeretnék képet adni a membrán GM1 gangliozidban gazdag mikrodoménjeinek szervező és moduláló funkciójáról néhány tumor sejtvonal, és azokat célzó molekuláris terápiák esetében. Kísérleteimben megvizsgáltam a tirozinkináz aktivitású PDGF receptorok (PDGFR) β típusának lipid-tutaj- és konfluenciafüggő szabályozását, továbbá feltártam a ciszplatin és egy új, még nem törzskönyvezett, igen hatékony platina alapú gyógyszerjelölt és a TRAIL halálligand raftfüggő szinergizmusát a DR4 és DR5 mediált, kaszpáz-8 dependens apoptózis indukciójában. Megállapítottam, hogy a sejtmembrán GM1 gazdag doménjei minden vizsgált rendszerben fontos szervezői a különböző útvonalakon lejátszódó jelátviteli folyamatoknak, és így nélkülözhetetlenek a vizsgált sejtfunkciók optimális működtetésében.ABSTRACT:In the last two decades, several publications dealt with the GM1-ganglioside rich domains of the cell membrane — also termed lipid rafts — as essential modulators of growth factor dependent signaling. These cholesterol- and sphingolipid-enriched submicron sized membrane domains possess not only structural, but also functional roles. Rafts are able to simultaneously isolate or accumulate diverse membrane proteins involved in various signaling pathways; furthermore, these platforms are also able to organize effector and adaptor proteins together with receptors in small membrane compartments, thus evoking more efficient signal transduction. In my PhD thesis I aim to demonstrate the modulatory and organizing function of GM1 rich microdomains in the membrane of tumor cell lines, and their possible role in targeted therapies. My observations encompass the significance of lipid rafts in serving as a platform for cell confluence dependent regulation of the functional outcome of signaling by the receptor tyrosine kinase PDGFR β, as well as for generating an apoptotic response to combination treatment with platinum complexes and TRAIL. Overall, my results support the view that GM1 rich microdomains are important regulators of various signaling pathways, and can be considered essential platforms for the optimal execution of cellular responses to external signals.N

A Small Number of HER2 Redirected CAR T Cells Significantly Improves Immune Response of Adoptively Transferred Mouse Lymphocytes against Human Breast Cancer Xenografts

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Enhanced endothelial motility and multicellular sprouting is mediated by the scaffold protein TKS4

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Trastuzumab derived HER2-specific CARs for the treatment of trastuzumab-resistant breast cancer: CAR T cells penetrate and eradicate tumors that are not accessible to antibodies

HER2-targeted monoclonal antibodies improve the outcome for advanced breast cancer patients; however, resistance to therapy is still frequent. Epitope masking and steric hindrance to antibody binding through matrix components are thought to be the major mechanism. We asked whether tumors resistant to trastuzumab can still be eliminated by CAR T cells redirected by the same antibody domain. While saturating doses of trastuzumab in the presence of CD16.176V.NK-92 effector cells and trastuzumab derived CAR T cells equally well recognized and killed HER2-positive tumor cells in a monolayer, only CAR T cells penetrated into the core region of tumor spheroids and exhibited cytotoxic activity in vitro, whereas antibodies failed. In NSG mice treatment with trastuzumab and CD16.176V.NK-92 cells only transiently retarded tumor growth but did not induce regression of clinically trastuzumab-resistant breast cancer xenografts. In contrast, one dose of HER2-specific CAR T cells eradicated established tumors resulting in long-term survival. Data indicate that CAR T cells can successfully combat antibody resistant tumors by targeting the same epitope suggesting that CAR T cells can penetrate the tumor matrix which is a barrier for antibodies

Cytolytic Activity of CAR T Cells and Maintenance of Their CD4+ Subset Is Critical for Optimal Antitumor Activity in Preclinical Solid Tumor Models

Correlative studies of clinical studies for hematological malignancies have implicated that less differentiated, CD8+-dominant CAR T cell products have greater antitumor activity. Here, we have investigated whether the differentiation status of CAR T cell products affects their antitumor activity in preclinical models of solid tumors. We explored if different activation/expansion protocols, as well as different co-stimulatory domains in the CAR construct, influence the short- and long-term efficacy of CAR T cells against HER2-positive tumors. We generated T cell products that range from the most differentiated (CD28.z; OKT3-antiCD28/RPMI expansion) to the least differentiated (41BB.z; OKT3-RetroNectin/LymphoONE expansion), as judged by cell surface expression of the differentiation markers CCR7 and CD45RA. While the effect of differentiation status was variable with regard to antigen-specific cytokine production, the most differentiated CD28.z CAR T cell products, which were enriched in effector memory T cells, had the greatest target-specific cytolytic activity in vitro. These products also had a greater proliferative capacity and maintained CD4+ T cells upon repeated stimulation in vitro. In vivo, differentiated CD28.z CAR T cells also had the greatest antitumor activity, resulting in complete response. Our results highlight that it is critical to optimize CAR T cell production and that optimal product characteristics might depend on the targeted antigen and/or cancer

CD28 and 41BB Costimulation Enhances the Effector Function of CD19-Specific Engager T Cells

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