scholarly journals Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Joshua D Greenlee ◽  
Maria Lopez-Cavestany ◽  
Nerymar Ortiz-Otero ◽  
Kevin Liu ◽  
Tejas Subramanian ◽  
...  

Colorectal cancer (CRC) remains a leading cause of cancer death, and its mortality is associated with metastasis and chemoresistance. We demonstrate that oxaliplatin-resistant CRC cells are sensitized to TRAIL-mediated apoptosis. Oxaliplatin-resistant cells exhibited transcriptional downregulation of caspase-10, but this had minimal effects on TRAIL sensitivity following CRISPR-Cas9 deletion of caspase-10 in parental cells. Sensitization effects in oxaliplatin-resistant cells were found to be a result of increased DR4, as well as significantly enhanced DR4 palmitoylation and translocation into lipid rafts. Raft perturbation via nystatin and resveratrol significantly altered DR4/raft colocalization and TRAIL sensitivity. Blood samples from metastatic CRC patients were treated with TRAIL liposomes, and a 57% reduction of viable circulating tumor cells (CTCs) was observed. Increased DR4/lipid raft colocalization in CTCs was found to correspond with increased oxaliplatin resistance and increased efficacy of TRAIL liposomes. To our knowledge, this is the first study to investigate the role of lipid rafts in primary CTCs.

2021 ◽  
Author(s):  
Joshua D. Greenlee ◽  
Maria Lopez Cavestany ◽  
Nerymar Ortiz-Otero ◽  
Kevin Liu ◽  
Tejas Subramanian ◽  
...  

Colorectal cancer (CRC) remains a leading cause of cancer death, and its mortality is associated with metastasis and resistance to chemotherapy. We demonstrate that oxaliplatin-resistant (OxR) CRC cells are sensitized to TRAIL-mediated apoptosis. Oxaliplatin-resistant cells exhibited transcriptional downregulation of caspase-10, but this effect was of little consequence to TRAIL sensitivity following CRISPR-Cas9 depletion of caspase-10. OxR cells were found to have increased expression of DR4, as well as significantly enhanced DR4 palmitoylation and translocation into lipid rafts. Raft perturbation via nystatin and resveratrol significantly altered DR4/raft colocalization and TRAIL sensitivity. Blood samples from metastatic CRC patients were treated with TRAIL liposomes, and a 57% reduction of viable circulating tumor cells (CTCs) was observed. The degree of DR4/lipid raft colocalization in CTCs was found to increase over time in patients receiving chemotherapy treatment. To our knowledge, this is the first study to investigate the role of lipid rafts in primary CTCs.


Oncotarget ◽  
2017 ◽  
Vol 8 (47) ◽  
pp. 82593-82608 ◽  
Author(s):  
Xian Zhang ◽  
Bo Feng ◽  
Fan Zhu ◽  
Chaoran Yu ◽  
Jiaoyang Lu ◽  
...  

2008 ◽  
Vol 28 (18) ◽  
pp. 5710-5723 ◽  
Author(s):  
Jia-Lin Lee ◽  
Mei-Jung Wang ◽  
Putty-Reddy Sudhir ◽  
Jeou-Yuan Chen

ABSTRACT CD44 is present in detergent-resistant, cholesterol-rich microdomains, called lipid rafts, in many types of cells. However, the functional significance of CD44 in lipid rafts is still unknown. We have previously demonstrated that osteopontin-mediated engagement of CD44 spliced variant isoforms promotes an extracellular matrix-derived survival signal through integrin activation. By using a series of CD44 mutants and pharmacological inhibitors selectively targeted to various cellular pathways, we show in this study that engagement of CD44 induces lipid raft coalescence to facilitate a CD44-Src-integrin signaling axis in lipid rafts, leading to increased matrix-derived survival. Palmitoylation of the membrane-proximal cysteine residues and carboxyl-terminal linkage to the actin cytoskeleton both contribute to raft targeting of CD44. The enrichment of integrin β1 in lipid rafts is tightly coupled to CD44 ligation-elicited lipid raft reorganization and associated with temporally delayed endocytosis. Through the interaction with the CD44 carboxyl-terminal ankyrin domain, Src is cotranslocated to lipid rafts, where it induces integrin activation via an inside-out mechanism. Collectively, this study demonstrates an important role of the dynamic raft reorganization induced by CD44 clustering in eliciting the matrix-derived survival signal.


Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3576-3576
Author(s):  
Patricia G. Quinter ◽  
Todd M. Quinton ◽  
Carol A. Dangelmaier ◽  
Satya P. Kunapuli ◽  
James L. Daniel

Abstract The collagen receptor glycoprotein VI (GPVI), plays an essential role in platelet activation and the regulation of hemostasis. Microdomains within the plasma membrane, called lipid rafts, have been implicated in GPVI signaling. The GPVI receptor has been shown to associate with the lipid rafts in both resting and activated platelets. It has been reported that there is a reduction in GPVI signaling in raft-disrupted platelets following activation with various GPVI agonists, especially at low to moderate agonist concentrations. Since platelet aggregation is potentiated by secreted adenosine 5′-diphosphate (ADP) at low concentrations of convulxin and at all concentrations of collagen and collagen-related peptide (CRP), we wanted to determine whether the decrease in GPVI signaling found in platelets with disrupted rafts was due to the loss of agonist potentiation by ADP. We compared platelet aggregation, protein phosphorylation, and calcium mobilization in platelets with intact and disrupted lipid rafts following activation with the GPVI agonists, collagen, convulxin and CRP. We show that lipid raft disruption inhibits aggregation induced by collagen and convulxin, but this inhibition is no longer apparent in the presence of ADP feedback inhibitors. Furthermore, raft-disrupted platelets had the same level of phosphorylation of proteins involved in GPVI signaling (i.e. Syk, LAT, and PLCγ2) and the same ability to mobilize calcium following activation with collagen or convulxin. Therefore, the effects of lipid raft disruption on aggregation can be attributed to the loss of ADP feedback. Interestingly, however, raft disruption directly inhibited aggregation and Syk phosphorylation induced by CRP in the presence and absence of ADP feedback. We propose that these differences are due to the fact that CRP is a relatively small, synthesized peptide of 37 amino acids, while collagen and convulxin are large ligands. These agonists are all able to bind the GPVI receptor, but they may not have the same ability to simultaneously cluster multiple receptors due to their size differential. The lipid rafts may be important for CRP stimulation, but not for collagen or convulxin, because they may have a higher density of the GPVI receptor than nonraft membrane regions, allowing CRP to cluster multiple receptors and activate the GPVI signaling cascade. When we disrupt the lipid rafts, we are reducing the effective concentration of GPVI available for activation by CRP but not by collagen or convulxin.


Bone ◽  
2011 ◽  
Vol 48 ◽  
pp. S255
Author(s):  
G. ¨Picarda ◽  
S. Surget ◽  
S. Télétchéa ◽  
M. Berreur ◽  
C. Pellat ◽  
...  

2019 ◽  
Author(s):  
Franziska Schmidt ◽  
Andreas Thywißen ◽  
Marie Röcker ◽  
Cristina Cunha ◽  
Zoltán Cseresnyés ◽  
...  

SUMMARYLipid rafts form signaling platforms on biological membranes with incompletely characterized role in immune response to infection. Here we report that lipid raft microdomains are essential components of the phagolysosomal membrane of macrophages. Genetic deletion of the lipidraft chaperons flotillin-1 and flotillin-2 demonstrate that the assembly of both major defense complexes vATPase and NADPH oxidase on the phagolysosomal membrane requires lipid rafts. Furthermore, we discovered a new virulence mechanism leading to the dysregulation of lipid-raft formation by melanized wild-type conidia of the important human-pathogenic fungusAspergillus fumigatus. This results in reduced phagolysosomal acidification. Phagolysosomes with ingested melanized conidia contain a reduced amount of free Ca2+ions as compared to phagolysosomes with melanin-free conidia. In agreement with a role of Ca2+for generation of functional lipid rafts, we show that Ca2+-dependent calmodulin activity is required for lipid-raft formation on the phagolysosome. We identified a single nucleotide polymorphism in the humanFLOT1gene that results in heightened susceptibility for invasive aspergillosis in hematopoietic stem-cell transplant recipients. Collectively, flotillin-dependent lipid rafts on the phagolysosomal membrane play an essential role in protective antifungal immunity in humans.


Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-3
Author(s):  
Magdalena Kucia ◽  
Kamila Bujko ◽  
Arjun Thapa ◽  
Janina Ratajczak ◽  
Mariusz Z Ratajczak

Background . It is known that prostaglandin E2 (PGE2) increases the homing and engraftment of hematopoietic stem/progenitor cells (HSPCs). However, aside from its role in upregulation of CXCR4 receptor expression on the surface of these cells, the exact mechanism has not been proposed. We have demonstrated in the past that an important step enabling the migration of HSPCs is the incorporation of CXCR4 into membrane lipid rafts on the leading surface (leading edge, in two dimensions) of migrating cells, which facilitates its interaction with cell migration signaling pathways (Wysoczynski M et al. Incorporation of CXCR4 into membrane lipid rafts primes homing-related responses of hematopoietic stem/progenitor cells to an SDF-1 gradient. Blood. 2005;105(1):40-48). Recently, we reported that Nlrp3 inflammasome-deficient HSPCs show a defect in lipid raft formation that results in defective migration of these cells in response to an SDF-1 gradient and their defective homing and engraftment after transplantation (Adamiak, M et al. Nlrp3 Inflammasome Signaling Regulates the Homing and Engraftment of Hematopoietic Stem Cells (HSPCs) by Enhancing Incorporation of CXCR4 Receptor into Membrane Lipid Rafts. Stem Cell Rev and Rep (2020). https://doi.org/10.1007/s12015-020-10005-w). An important activator of Nlrp3 inflammasomes is reactive oxygen species (ROS). Importantly, the enzyme that generates ROS, known as NADPH oxidase 2 (NOX2), is also associated with cell membrane lipid rafts. Hypothesis. Given the known roles of PGE2, membrane lipid rafts, and the Nlrp3 inflammasome in migration, homing, and engraftment of HSPCs, we hypothesized that PGE2 signaling promotes Nlrp3 inflammasome activation in a Nox2-ROS-dependent manner that results in incorporation of CXCR4 into membrane lipid rafts, which better explains the role of PGE2 in these phenomena.Materials and Methods. To test this hypothesis, murine SKL and human CD34+ cells enriched for HSPCs were stimulated with PGE2 to evaluate activation of genes of the Nlrp3 inflammasome complex at the mRNA and protein levels. Next, HSPCs from Nox2-KO mice were tested for membrane lipid raft formation in functional chemotaxis assays in response to SDF-1 gradients under conditions promoting membrane lipid raft formation. Formation of membrane lipid rafts in Nox2-KO cells was also evaluated by confocal analysis in the presence or absence of PGE2. Finally, the effect of the PGE2-Nox2-Nlrp3 inflammasome axis on the formation of membrane lipid rafts was evaluated in the presence of the ROS scavenger N-acethyl-cysteine (NAC). Results. We provide for the first time evidence that PGE2 activates Nlrp3 inflammasomes in HSPCs in a Nox2-ROS-dependent manner. This Nlrp3 inflammasome activation increases at the leading surface of migrating HSPCs with incorporation of the CXCR4 receptor into membrane lipid rafts. Formation of membrane lipid rafts was absent in Nox2-KO and Nlrp3-KO mouse HSPCs and in normal wild type cells after their exposure to NAC. Moreover, we also observed that Nox2-KO and Nlrp3-KO mice had a lower basal level of CXCR4 expression. Conclusions. Our results for the first time explain the role of PGE2 in promoting homing and migration of HSPCs, which occurs in response to PGE2 by activation of the Nox2-ROS-Nlrp3 inflammasome axis and thereby promotes incorporation of the CXCR4 receptor into membrane lipid rafts. Moreover, basal expression of the CXCR4 receptor was at a low level on the surface of HSPCs from Nlrp3-KO mice. Thus, our results provide evidence for the importance of the Nox2-ROS-Nlrp3 inflammasome axis in PGE2-mediated homing and engraftment of HSPCs and the role of PGE2-mediated lipid raft formation for optimal responsiveness of CXCR4 to SDF-1 in the BM microenvironment. Disclosures No relevant conflicts of interest to declare.


Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4248-4248
Author(s):  
Yoko Tabe ◽  
Linhua Jin ◽  
Yixin Zhou ◽  
Naoki Ichikawa ◽  
Kazuhisa Iwabuchi ◽  
...  

Abstract Abstract 4248 In patients with chronic-phase chronic myeloid leukemia (CML), imatinib resistance is of increasing importance. We have recently reported that the constitutively activated Bcr-Abl tyrosine kinase in CML suppresses CXCL12/CXCR4-mediated migration of CML cells to the bone marrow (BM) stroma. This finding can explain the characteristic leukocytosis in CML. In turn, tyrosine kinase inhibitor imatinib inhibits Bcr-Abl, enhances migration of CML cells towards CXCL12-producing BM stromal cells which in turn promotes cell quiescence and development of the microenvironment-mediated, non-pharmacological drug resistance (Jin, Mol Cancer Ther 2008;7:48). In this study, we further investigated the molecular mechanisms of imatinib-induced CML cell migration and adherence to the bone marrow-derived stromal cells (MSC). Src-related kinase Lyn regulates survival and responsiveness of CML cells to inhibition of BCR-ABL kinase and is known to interact with CXCL12/CXCR4 signaling. Lyn frequently localizes in lipid raft fractions, which act as signal transduction platforms for a variety of intracellular processes. Therefore, we investigated the effects of imatinib on the localization of activated Lyn in the lipid raft structures of KBM-5 CML cells under co-culture conditions with CXCL12-secreting MSC or recombinant CXCL12. Confocal microscopy and discontinuous sucrose density gradient fractionation demonstrated that CXCR4 and phosphorylated CXCR4 localized in the higher-density detergent-soluble non-raft cell surface regions in KBM5 cells in the presence and absence of imatinib, with or without MSC, which suggests that CXCR4 does not directly associate with lipid rafts. In contrast, Lyn was present both in the low-density raft and in the high-density non-raft fractions, which contained CXCR4. We have further demonstrated co-localization of CXCR4 with Lyn, and their direct interaction was confirmed by co-immunoprecipitation. Notably, the active form of phosphorylated p-LynTyr396 clustered in lipid rafts, while inactive p-LynTyr507 in non-raft fractions. In suspension KBM-5 cultures imatinib depleted both, p-LynTyr396 and p-LynTyr507. In contrast, under MSC co-culture conditions imatinib repressed p-LynTyr507, but failed to deplete p-LynTyr396 in lipid rafts, and p-LynTyr396 further accumulated in non-raft fractions, likely associating with CXCR4. Knock-down of Lyn by siRNA, Src inhibitor treatment or lipid raft destruction by methyl-b cyclodextrin (MbCD) abrogated imatinib-induced KBM5 migration to MSCs and CXCL12, indicating the critical role of p-LynTyr396 in cell migration. Since the a4b1 integrin VLA-4 represents a cooperative molecular pathway guiding BM homing in addition to CXCL12/CXCR4, we next investigated the localization and expression of VLA-4 in KBM5 cells. Imatinib decreased VLA-4 protein expression both in lipid raft and non-raft fractions without affecting VLA-4 gene expression levels as determined by quantitative RT-PCR. Interestingly, VLA-4 reduction by imatinib or lipid raft destruction by MbCD did not affect the ability to adhere to fibronectin. In conclusion, these findings demonstrate that under conditions mimicking BM microenvironment imatinib restores CXCL12-dependent migration through interactions between CXCR4 and active p-Lyn Tyr396 in non-raft microdomains of CML cells and that p-Lyn Tyr396 localized in lipid rafts is contributing to the CML cell migration. We propose that while CXCR4 is segregated from lipid raft fractions, imatinib through Bcr-Abl kinase inhibition induces the compartmental changes of multivalent Lyn complex between lipid raft and non-raft fractions, restoring the interactions between Lyn and CXCR4 and stimulating cell migration. Our findings indicate that leukemic BM microenvironment may be involved in imatinib resistance in a subset of CML patients through activation of Lyn kinase, consistent with reported higher clinical activity of Bcr-Abl/Src inhibitor dasatinib in patients with imatinib-resistant CML. We propose that BM stroma cells produce abundant CXCL12 and attract migrating cells through adhesive interactions with the extracellular matrix, which may in turn facilitate lodging into BM niches of imatinib-exposed CML cells and promote non-pharmacological resistance to this agent. Disclosures: No relevant conflicts of interest to declare.


2004 ◽  
Vol 32 (5) ◽  
pp. 679-681 ◽  
Author(s):  
D. Scheel-Toellner ◽  
K. Wang ◽  
L.K. Assi ◽  
P.R. Webb ◽  
R.M. Craddock ◽  
...  

Neutrophils die by apoptosis spontaneously within 12–24 h of their release from the bone marrow. The mechanism regulating entry of neutrophils into apoptosis at the end of their life-span is currently under debate. Our data suggest that neutrophil apoptosis involves a novel mechanism of caspase 8 activation that is indirectly regulated by accumulation of reactive oxygen species. We detected early activation of caspase 8 upstream of caspase 3 activation, suggesting death receptor signalling. The CD95 DISC (death-inducing signalling complex) was detected in neutrophils, but blocking antibodies to death receptors did not inhibit apoptosis, suggesting a novel mechanism for caspase 8 activation. Death receptor clustering in ceramide-rich lipid rafts is thought to be an early event in their signalling, so we investigated the role of ceramide generated by ASM (acid sphingomyelinase) in neutrophil apoptosis. Ceramide was generated early in neutrophil apoptosis, and ASM activity was required for neutrophil apoptosis. Moreover, neutrophil apoptosis was significantly delayed in ASM−/− mice compared with their wild-type littermates. CD95 DISC components were present in lipid rafts in neutrophils, and were progressively clustered in cultured neutrophils. Generation of ceramide was blocked by desferrioxamine, suggesting that hydroxyl radicals are important for the activation of ASM. This observation was in line with our earlier observation of a precipitous drop in reduced glutathione in the aging neutrophil.


2022 ◽  
Vol 23 (1) ◽  
pp. 533
Author(s):  
Karel Paukner ◽  
Ivana Králová Lesná ◽  
Rudolf Poledne

Membrane cholesterol is essential for cell membrane properties, just as serum cholesterol is important for the transport of molecules between organs. This review focuses on cholesterol transport between lipoproteins and lipid rafts on the surface of macrophages. Recent studies exploring this mechanism and recognition of the central dogma—the key role of macrophages in cardiovascular disease—have led to the notion that this transport mechanism plays a major role in the pathogenesis of atherosclerosis. The exact molecular mechanism of this transport remains unclear. Future research will improve our understanding of the molecular and cellular bases of lipid raft-associated cholesterol transport.


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