Lipid rafts in cancer chemotherapy

Trabajo presentado por Mariana Reis Sobreiro para optar al grado de Doctor por la Universidad de Salamanca y realizado en el Instituto de Biología Molecular y Celular del Cáncer.[ES]: La continua proliferación de las células tumorales implica una gran demanda de síntesis de nuevas membranas. Ya al principio del siglo 20 se observó una mayor acumulación de colesterol en tumores y en los tejidos adyacentes. Se comenzó a discutir la importancia del colesterol en la progresión tumoral cuando se observó una acumulación irregular en células malignas y su entorno, consecuencia de la alta tasa de proliferación de las células cancerosas, conllevando a una gran demanda de síntesis de nuevas membranas. El descubrimiento de la existencia de microdominios de membrana lipid raft en células normales, ricos en colesterol y especializados en señalización, llevo al estudio del papel de estos dominios en procesos aberrantes y patológicos como la oncogénesis y de metástasis. Estudios recientes demuestran que los dominios de membrana raft están implicados en la agresividad y crecimiento del tumor. Se ha descrito una nueva clase de compuestos denominados lípidos antitumorales (ATLs), los más conocidos perifosina y edelfosina, capaces de inducir apoptosis selectivamente en células malignas mediante un proceso dependiente de microdominios de membrana rafts. Estudios biofísicos han demostrado que el ATL edelfosina posee afinidad por colesterol. Estos compuestos se acumulan en microdominios rafts de células malignas activando la apoptosis por movilización del receptor de muerte Fas/CD95 hacia dominios rafts, de forma independiente de su ligando natural FasL/CD95L, y reclutando proteínas señalizadoras de apoptosis downstream. La incubación con ATLs activa, mediante la reorganización de los dominios de membrana rafts en células tumorales, las vías apoptóticas extrínseca (receptores de muerte) e intrínseca (mitocondrial), vías que se encuentran generalmente bloqueadas en células tumorales e inhibe la activación de la ruta PI3K/Akt, dependiente de la integridad de los raft. Asímismo, se ha descrito que en tumores hay reclutamiento de Fas/CD95 en lipid rafts por compuestos que son químicamente diferentes a los ATLs, como el cisplatino, resveratrol, entre otros, evidenciando que este mecanismo es más general de lo que se creía inicialmente. Algunos estudios sugieren la posibilidad de que este fenómeno sea una consecuencia de la inhibición de PI3K/Akt, conllevando a la activación de Fas/CD95 en lipid rafts, por un mecanismo todavía desconocido.[EN]: The ongoing proliferation of tumor cells involves a great demand for synthesis of new membranes . Since the early 20th century increased cholesterol accumulation in tumors and adjacent tissues was observed. He began to discuss the importance of cholesterol in tumor progression when an irregular accumulation in malignant cells and their environment due to the high rate of proliferation of cancer cells , leading to a high demand for synthesis of new membranes was observed. The discovery of the existence of lipid raft membrane microdomains in normal cells , high cholesterol and specialized in signage, led to the study of the role of these domains in aberrant and pathological processes such as oncogenesis and metastasis. Recent studies show that membrane raft domains are involved in tumor growth and aggressiveness . Described a new class of antitumor compounds termed lipids ( ATLs ) , the best known perifosine and edelfosine , capable of inducing apoptosis selectively in cancer cells by a process dependent membrane microdomains rafts . Biophysical studies have shown that the affinity has edelfosine ATL cholesterol . These compounds accumulate in malignant cell rafts microdomains activating apoptosis death receptor mobilization Fas/CD95 to rafts domains independently of its natural ligand FasL/CD95L and apoptosis by recruiting downstream signaling proteins . Incubation with active ATLs through reorganization of membrane rafts domains on tumor cells, the extrinsic apoptotic pathways (death receptor ) and intrinsic (mitochondrial) , pathways are typically locked in tumor cells and inhibits the activation of the path PI3K/Akt dependent raft integrity.This thesis was financed by Ministério da Ciência, Tecnologia e Ensino Superior, predoctoral fellowship, Fundação para a Ciência e a Tecnologia (FCT), Portugal.Peer Reviewe

Involvement of mitochondria and recruitment of Fas/CD95 signaling in lipid rafts in resveratrol-mediated antimyeloma and antileukemia actions

We have found that resveratrol (trans-3,4′,5-trihydroxystilbene) induced apoptosis in multiple myeloma (MM) and T-cell leukemia cells through coclustering of Fas/CD95 death receptor and lipid rafts, whereas normal lymphocytes were spared. Tumor necrosis factor-related apoptosis-inducing ligand receptors, Fas-associated death domain-containing protein (FADD), procaspase-8, procaspase-10, c-Jun amino-terminal kinase and Bid were also recruited into lipid rafts on resveratrol incubation with MM and T-cell leukemia cells. Raft disruption inhibited resveratrol-induced apoptosis. Bcl-X L overexpression prevented resveratrol-induced disruption of mitochondrial transmembrane potential (Δψ m) and apoptosis. A FADD dominant-negative mutant, that blocked Fas/CD95 downstream signaling, precluded resveratrol-induced Δψ m loss and apoptosis, indicating a sequence of Fas/CD95 signalingmitochondrion in the apoptotic response triggered by resveratrol. Cells deficient in Fas/CD95 did not undergo resveratrol-induced apoptosis. Pretreatment of MM cells with interferon-γ upregulated Fas/CD95 and caspase-8, and potentiated resveratrol-induced apoptosis. Our data indicate that recruitment of Fas/CD95 death receptor and downstream signaling molecules into lipid rafts, followed by Δψ m disruption, underlies the apoptotic action of resveratrol in MM and T-cell leukemic cells. Combination of resveratrol with perifosine or bortezomib potentiated the apoptotic response induced by each single drug. These results also highlight the role of recruitment of Fas/CD95 signaling in lipid rafts in antimyeloma and antileukemia chemotherapy.This work was supported by grants from Ministerio de Ciencia e Innovación (SAF2008-02251, SAF2005-04293, RD06/0020/ 1037 – Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III), Fondo de Investigación Sanitaria and European Commission (FIS-FEDER 06/0813, 04/0843), Junta de Castilla y León (CSI01A08, SAN673/SA32/ 08, and GR15-Experimental Therapeutics and Translational Oncology Program), Fundación de Investigación Médica Mutua Madrileña (FMM) and Fundación ‘la Caixa’ (BM05- 30-0). MRS is recipient of a predoctoral fellowship from the Fundaçâo para a Ciencia e Tecnologia (Ministério da Ciencia, Tecnología e Ensino Superior of Portugal). CG is supported by the Ramón y Cajal Program from the Ministerio de Ciencia e Innovación of Spain.Peer Reviewe

Lipid raft-mediated Akt signaling as a therapeutic target in mantle cell lymphoma

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.Recent evidence shows that lipid raft membrane domains modulate both cell survival and death. Here, we have found that the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is present in the lipid rafts of mantle cell lymphoma (MCL) cells, and this location seems to be critical for full activation and MCL cell survival. The antitumor lipids (ATLs) edelfosine and perifosine target rafts, and we found that ATLs exerted in vitro and in vivo antitumor activity against MCL cells by displacing Akt as well as key regulatory kinases p-PDK1 (phosphatidylinositol-dependent protein kinase 1), PI3K and mTOR (mammalian TOR) from lipid rafts. This raft reorganization led to Akt dephosphorylation, while proapoptotic Fas/CD95 death receptor was recruited into rafts. Raft integrity was critical for Ser473 Akt phosphorylation. ATL-induced apoptosis appeared to correlate with the basal Akt phosphorylation status in MCL cell lines and primary cultures, and could be potentiated by the PI3K inhibitor wortmannin, or inhibited by the Akt activator pervanadate. Classical Akt inhibitors induced apoptosis in MCL cells. Microenvironmental stimuli, such as CD40 ligation or stromal cell contact, did not prevent ATL-induced apoptosis in MCL cell lines and patient-derived cells. These results highlight the role of raft-mediated PI3K/Akt signaling in MCL cell survival and chemotherapy, thus becoming a new target for MCL treatment. © 2013 Macmillan Publishers Limited.This work was supported by grants from Ministerio de Economía y Competitividad of Spain (SAF2008-02251 and SAF2011-30518 to FM; SAF2009-09503 to DC), Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, cofunded by the Fondo Europeo de Desarrollo Regional of the European Union (RD06/0020/1037 and RD12/0036/0065 to FM; RD06/0020/0014 to DC), Fondo de Investigación Sanitaria and European Commission, Instituto de Salud Carlos III (PI09/0060 to GR; PS09/01915 to CG), European Community’s Seventh Framework Programme FP7-2007-2013 (grant HEALTH-F2-2011-256986, PANACREAS to FM), Junta de Castilla y León (CSI052A11-2 and CSI221A12-2 to FM; Biomedicine Project 2010–2011 to CG) and Generalitat de Catalunya (2009SGR967 to DC). MRS was recipient of a predoctoral fellowship from the Fundaçâo para a Ciência e Tecnologia (Ministério da Ciencia, Tecnología e Ensino Superior of Portugal), and AM was a recipient of a predoctoral fellowship from IDIBAPS. CG was supported by the Ramón y Cajal Program from the Ministerio de Ciencia e Innovación of Spain.Peer Reviewe

Caenorhabditis elegans as a platform to study the mechanism of action of synthetic antitumor lipids

Drugs capable of specifically recognizing and killing cancer cells while sparing healthy cells are of great interest in anti-cancer therapy. An example of such a drug is edelfosine, the prototype molecule of a family of synthetic lipids collectively known as antitumor lipids (ATLs). A better understanding of the selectivity and the mechanism of action of these compounds would lead to better anticancer treatments. Using Caenorhabditis elegans, we modeled key features of the ATL selectivity against cancer cells. Edelfosine induced a selective and direct killing action on C. elegans embryos, which was dependent on cholesterol, without affecting adult worms and larvae. Distinct ATLs ranked differently in their embryonic lethal effect with edelfosine > perifosine > erucylphosphocholine >> miltefosine. Following a biased screening of 57 C. elegans mutants we found that inactivation of components of the insulin/IGF-1 signaling pathway led to resistance against the ATL edelfosine in both C. elegans and human tumor cells. This paper shows that C. elegans can be used as a rapid platform to facilitate ATL research and to further understand the mechanism of action of edelfosine and other synthetic ATLs.This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación (SAF2011–30518), Spanish Ministerio de Economía y Competitividad (RD12/0036/0065, Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, cofunded by the Fondo Europeo de Desarrollo Regional of the European Union), European Community’s Seventh Framework Program FP7-2007-2013 (grant HEALTH-F2–2011–256986, PANACREAS), and Junta de Castilla y León (CSI052A11–2). ASB was supported by the CSIC JAE-Doc program.Peer Reviewe

Saracatinib impairs maintenance of human T-ALL by targeting the LCK tyrosine kinase in cells displaying high level of lipid rafts

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Lipid raft isolation by sucrose gradient centrifugation and visualization of raft-located proteins by fluorescence microscopy: the use of combined techniques to assess Fas/CD95 location in rafts during apoptosis triggering

40 p.-11 fig.-6 tab.Lipid rafts are heterogeneous membrane domains enriched in cholesterol, sphingolipids, and gangliosides that serve as sorting platforms to compartmentalize and modulate signaling pathways. Death receptors and downstream signaling molecules have been reported to be recruited into these raft domains during the triggering of apoptosis. Here, we provide two protocols that support the presence of Fas/CD95 in lipid rafts during apoptosis, involving lipid raft isolation and confocal microscopy techniques. A detailed protocol is provided for the isolation of lipid rafts, by taking advantage of their resistance to Triton X-100 solubilization at 4 °C, followed by subsequent sucrose gradient centrifugation and analysis of the protein composition of the different gradient fractions by Western blotting. In addition, we also provide a detailed protocol for the visualization of the coclustering of Fas/CD95 death receptor and lipid rafts, as assessed by using anti-Fas/CD95 antibodies and fluorescent dye-conjugated cholera toxin B subunit that binds to ganglioside GM1, a main component of lipid rafts, by immunofluorescence and confocal microscopy. These protocols can be extended to any protein of interest to be analyzed for its association to lipid rafts.This work was supported by Spanish Ministry of Science, Innovation and Universities grant SAF2017-89672-R.Peer reviewe