Increased Plasma Membrane Fluidity Research Articles

Cellular and Molecular Processes Are Differently Influenced in Primary Neural Cells by Slight Changes in the Physicochemical Properties of Multicore Magnetic Nanoparticles

Herein, we use two exemplary superparamagnetic iron oxide multicore nanoparticles (SPIONs) to illustrate the significant influence of slightly different physicochemical properties on the cellular and molecular processes that define SPION interplay with primary neural cells. Particularly, we have designed two different SPION structures, NFA (i.e., a denser multicore structure accompanied by a slightly less negative surface charge and a higher magnetic response) and NFD (i.e., a larger surface area and more negatively charged), and identified specific biological responses dependent on SPION type, concentration, exposure time, and magnetic actuation. Interestingly, NFA SPIONs display a higher cell uptake, likely driven by their less negative surface and smaller protein corona, more significantly impacting cell viability and complexity. The tight contact of both SPIONs with neural cell membranes results in the significant augmentation of phosphatidylcholine, phosphatidylserine, and sphingomyelin and the reduction of free fatty acids and triacylglycerides for both SPIONs. Nonetheless, NFD induces greater effects on lipids, especially under magnetic actuation, likely indicating a preferential membranal location and/or a tighter interaction with membrane lipids than NFA, in agreement with their lower cell uptake. From a functional perspective, these lipid changes correlate with an increase in plasma membrane fluidity, again larger for more negatively charged nanoparticles (NFD). Finally, the mRNA expression of iron-related genes such as Ireb-2 and Fth-1 remains unaltered, while TfR-1 is only detected in SPION-treated cells. Taken together, these results demonstrate the substantial impact that minor physicochemical differences of nanomaterials may exert in the specific targeting of cellular and molecular processes. A denser multicore structure generated by autoclave-based production is accompanied by a slight difference in surface charge and magnetic properties that become decisive for the biological impact of these SPIONs. Their capacity to markedly modify the lipidic cell content makes them attractive as lipid-targetable nanomedicines.

In vitro aging of stallion spermatozoa during prolonged storage at 5°C.

Artificial insemination with chilled stallion semen is hampered by a limited period of maximum fertility maintenance (24-48 h). This study used multiparametric flow cytometry to simultaneously measure reactive oxygen species (ROS) production, mitochondrial function or [Ca2+ ]i and plasma membrane fluidity in viable, acrosome-intact spermatozoa, with the aim of providing insight into changes in sperm function during storage at 5°C. High proportions of viable and acrosome-intact spermatozoa (71 ± 8%) remained after 96 h of storage demonstrating that the basic integrity of the cells was well preserved (n=17 stallions). In addition, more than 90% of viable, acrosome-intact spermatozoa had active mitochondria and low intra-cellular or mitochondrial ROS levels. By contrast, the percentage of viable, acrosome-intact sperm with low plasma membrane fluidity and low [Ca2+ ]i decreased over time (1h: 63 ± 16%, 96 h: 29 ± 18%; p < 0.05). The [Ca2+ ]i in viable sperm rose 3.1-fold (p < 0.05) over the 4 days, and fewer spermatozoa responded to bicarbonate stimulation (1h: 46 ± 17%, 96 h: 19 ± 12%) with an increase in plasma membrane fluidity following prolonged storage. Overall, prolonged storage of stallion semen at 5°C resulted in disturbed calcium homeostasis and increased plasma membrane fluidity. The decline in fertility of stallion semen during cooled-storage may therefore relate to aspects of in vitro aging (changes in plasma membrane fluidity and intracellular calcium) which impairs capacitation-associated cell functions.

NSCLC Cells Resistance to PI3K/mTOR Inhibitors Is Mediated by Delta-6 Fatty Acid Desaturase (FADS2)

Hyperactivation of the phosphatidylinositol-3-kinase (PI3K) pathway is one of the most common events in human cancers. Several efforts have been made toward the identification of selective PI3K pathway inhibitors. However, the success of these molecules has been partially limited due to unexpected toxicities, the selection of potentially responsive patients, and intrinsic resistance to treatments. Metabolic alterations are intimately linked to drug resistance; altered metabolic pathways can help cancer cells adapt to continuous drug exposure and develop resistant phenotypes. Here we report the metabolic alterations underlying the non-small cell lung cancer (NSCLC) cell lines resistant to the usual PI3K-mTOR inhibitor BEZ235. In this study, we identified that an increased unsaturation degree of lipid species is associated with increased plasma membrane fluidity in cells with the resistant phenotype and that fatty acid desaturase FADS2 mediates the acquisition of chemoresistance. Therefore, new studies focused on reversing drug resistance based on membrane lipid modifications should consider the contribution of desaturase activity.

Abstract 2163: Gender and estrogens as key factors in the response to immunotherapy in non small cell lung cancer

Abstract Gender is an important stratification and prognostic factor in oncology in the response to treatments, particularly to the immunotherapy. The anti-PD-1/PD-L1 immune-checkpoint inhibitors (Pembrolizumab, Nivolumab, Atezolizumab) have improved the prognosis of non-small cell lung cancer (NSCLC) patients in 20-35% cases, but with a lower efficacy in females. The different steroid hormones - estrogens, progesterone or androgens - present in both gender have often contrasting effects on the activity of the host immune system, partly explaining the differences observed. It is not known if metabolic features of cancer cells of male and female patients may impact on the immune system response and immunotherapy efficacy. We set up an analytical pipeline based on patient-derived samples, cell lines and humanized male and female mice bearing gender-matched NSCLC xenografts. NanoString analysis in a cohort of 42 NSCLC patients of both genders treated with Pembrolizumab, identified the estrogen receptor α (ERα)/ESR1 gene as one of the most significantly associated with the response to Pembrolizumab, particularly in female patients. A panel of 30 human NSCLC cell lines of female and male origin, that reproduced the ERα/gender association observed in clinical samples, the amount of ERα and 17-β-estradiol was directly related to the expression of PD-L1, suggesting a potential direct linkage. ERα transcriptionally up-regulated CD274/PD-L1 gene, with higher effects in females. EGFR downstream effectors Akt and ERK1/2, increased the active form phospho(Ser118)ERα, upregulating PD-L1. Inhibiting the estrogen synthesis or switching off EGFR activity down-regulated PD-L1 levels. Consistently, the aromatase inhibitor letrozole increased the efficacy of Pembrolizumab in humanized NSCLC xenografts, by reducing tumor growth and tumor-associated PD-L1, increasing the infiltrating CD8+T-lymphocytes, NK cells and Vγ9Vδ2 anti-tumor T-lymphocytes. The benefit was maximal in 17-β-estradiol/ERα high female xenografts, intermediate in 17-β-estradiol/ERα medium female and male xenografts, minimal in 17-β-estradiol/ERα low male xenografts. Additionally, an untargeted lipidome analysis revealed that letrozole decreased the saturated fatty acids (SFAs)/mono- or poly-unsaturated fatty acids (MUFAs-PUFAs) ratio, and the cholesterol/cholesterol esters ratio. These changes increased plasma-membrane fluidity, an event that in turns reduced the affinity between PD-L1 and PD-1. As further proof of concept, ω6- or ω3-PUFA treated cells, displayed an increased membrane fluidity and a change in lipid metabolism that affect the response to anti-PD-1/PD-L1 as letrozole did. We propose a prototypical in vitro 3D spheroids culture gender-tailored platform that allows to investigate the metabolic circuitries causing such differences and to repurpose metabolic modifiers as novel immune-adjuvant agents in oncology. Citation Format: Sofia La Vecchia, Dario Pasquale Anobile, Fabrizio Tabbò, Iris C. Salaroglio, Joanna Kopecka, Luca Primo, Luisella Righi, Giorgio Vittorio Scagliotti, Silvia Novello, Cyril Corbet, Chiara Riganti. Gender and estrogens as key factors in the response to immunotherapy in non small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2163.

Bicarbonate-Stimulated Membrane Reorganization in Stallion Spermatozoa.

Classical in vitro fertilization (IVF) is still poorly successful in horses. This lack of success is thought to be due primarily to inadequate capacitation of stallion spermatozoa under in vitro conditions. In species in which IVF is successful, bicarbonate, calcium, and albumin are considered the key components that enable a gradual reorganization of the sperm plasma membrane that allows the spermatozoa to undergo an acrosome reaction and fertilize the oocyte. The aim of this work was to comprehensively examine contributors to stallion sperm capacitation by investigating bicarbonate-induced membrane remodelling steps, and elucidating the contribution of cAMP signalling to these events. In the presence of capacitating media containing bicarbonate, a significant increase in plasma membrane fluidity was readily detected using merocyanine 540 staining in the majority of viable spermatozoa within 15 min of bicarbonate exposure. Specific inhibition of soluble adenylyl cyclase (sAC) in the presence of bicarbonate by LRE1 significantly reduced the number of viable sperm with high membrane fluidity. This suggests a vital role for sAC-mediated cAMP production in the regulation of membrane fluidity. Cryo-electron tomography of viable cells with high membrane fluidity revealed a range of membrane remodelling intermediates, including destabilized membranes and zones with close apposition of the plasma membrane and the outer acrosomal membrane. However, lipidomic analysis of equivalent viable spermatozoa with high membrane fluidity demonstrated that this phenomenon was neither accompanied by a gross change in the phospholipid composition of stallion sperm membranes nor detectable sterol efflux (p > 0.05). After an early increase in membrane fluidity, a significant and cAMP-dependent increase in viable sperm with phosphatidylserine (PS), but not phosphatidylethanolamine (PE) exposure was noted. While the events observed partly resemble findings from the in vitro capacitation of sperm from other mammalian species, the lack of cholesterol removal appears to be an equine-specific phenomenon. This research will assist in the development of a defined medium for the capacitation of stallion sperm and will facilitate progress toward a functional IVF protocol for horse gametes.

Unexpected Role of Sterol Synthesis in RNA Stability and Translation in Leishmania.

Leishmania parasites are trypanosomatid protozoans that cause leishmaniasis affecting millions of people worldwide. Sterols are important components of the plasma and organellar membranes. They also serve as precursors for the synthesis of signaling molecules. Unlike animals, Leishmania does not synthesize cholesterol but makes ergostane-based sterols instead. C-14-demethylase is a key enzyme involved in the biosynthesis of sterols and an important drug target. In Leishmania parasites, the inactivation of C-14-demethylase leads to multiple defects, including increased plasma membrane fluidity, mitochondrion dysfunction, hypersensitivity to stress and reduced virulence. In this study, we revealed a novel role for sterol synthesis in the maintenance of RNA stability and translation. Sterol alteration in C-14-demethylase knockout mutant leads to increased RNA degradation, reduced translation and impaired heat shock response. Thus, sterol biosynthesis in Leishmania plays an unexpected role in global gene regulation.

Stable reduction of STARD4 alters cholesterol regulation and lipid homeostasis

STARD4, a member of the evolutionarily conserved START gene family, is a soluble sterol transport protein implicated in cholesterol sensing and maintenance of cellular homeostasis. STARD4 is widely expressed and has been shown to transfer sterol between liposomes as well as organelles in cells. However, STARD4 knockout mice lack an obvious phenotype, so the overall role of STARD4 is unclear. To model long term depletion of STARD4 in cells, we use short hairpin RNA technology to stably decrease STARD4 expression in human U2OS osteosarcoma cells (STARD4-KD). We show that STARD4-KD cells display increased total cholesterol, slower cholesterol trafficking between the plasma membrane and the endocytic recycling compartment, and increased plasma membrane fluidity. These effects can all be rescued by transient expression of a short hairpin RNA-resistant STARD4 construct. Some of the cholesterol increase was due to excess storage in late endosomes or lysosomes. To understand the effects of reduced STARD4, we carried out transcriptional and lipidomic profiling of control and STARD4-KD cells. Reduction of STARD4 activates compensatory mechanisms that alter membrane composition and lipid homeostasis. Based on these observations, we propose that STARD4 functions as a critical sterol transport protein involved in sterol sensing and maintaining lipid homeostasis.

Plant-Unique cis/trans Isomerism of Long-Chain Base Unsaturation is Selectively Required for Aluminum Tolerance Resulting from Glucosylceramide-Dependent Plasma Membrane Fluidity.

Cis/trans isomerism of the Δ8 unsaturation of long-chain base (LCB) is found only in plant sphingolipids. This unique geometry is generated by sphingolipid LCB Δ8 desaturase SLD which produces both isomers at various ratios, resulting in diverse cis/trans ratios in plants. However, the biological significance of this isomeric diversity remains controversial. Here, we show that the plant-specific cis unsaturation of LCB selectively contributes to glucosylceramide (GlcCer)-dependent tolerance to aluminum toxicity. We established three transgenic rice lines with altered LCB unsaturation profiles. Overexpression of SLD from rice (OsSLD-OX), which preferentially exhibits cis-activity, or Arabidopsis (AtSLD-OX), showing preference for trans-activity, facilitated Δ8 unsaturation in different manners: a slight increase of cis-unsaturated glycosylinositolphosphoceramide (GIPC) in OsSLD-OX, and a drastic increase of trans-unsaturated GlcCer and GIPC in AtSLD-OX. Disruption of LCB Δ4 desaturase (des) significantly decreased the content of GlcCer. Fluorescence imaging analysis revealed that OsSLD-OX and AtSLD-OX showed increased plasma membrane fluidity, whereas des had less fluidity, demonstrating that the isomers universally contributed to increasing membrane fluidity. However, the results of a hydroponic assay showed decreased aluminum tolerance in AtSLD-OX and des compared to OsSLD-OX and the control plants, which did not correlate with membrane fluidity. These results suggest that cis-unsaturated GlcCer, not GIPC, selectively serves to maintain the membrane fluidity specifically associated with aluminum tolerance.

StarD5: an ER stress protein regulates plasma membrane and intracellular cholesterol homeostasis

How plasma membrane (PM) cholesterol is controlled is poorly understood. Ablation of the gene encoding the ER stress steroidogenic acute regulatory-related lipid transfer domain (StarD)5 leads to a decrease in PM cholesterol content, a decrease in cholesterol efflux, and an increase in intracellular neutral lipid accumulation in macrophages, the major cell type that expresses StarD5. ER stress increases StarD5 expression in mouse hepatocytes, which results in an increase in accessible PM cholesterol in WT but not in StarD5-/- hepatocytes. StarD5-/- mice store higher levels of cholesterol and triglycerides, which leads to altered expression of cholesterol-regulated genes. In vitro, a recombinant GST-StarD5 protein transfers cholesterol between synthetic liposomes. StarD5 overexpression leads to a marked increase in PM cholesterol. Phasor analysis of 6-dodecanoyl-2-dimethylaminonaphthalene fluorescence lifetime imaging microscopy data revealed an increase in PM fluidity in StarD5-/- macrophages. Taken together, these studies show that StarD5 is a stress-responsive protein that regulates PM cholesterol and intracellular cholesterol homeostasis.

Exposure to a Nonionic Surfactant Induces a Response Akin to Heat-Shock Apoptosis in Intestinal Epithelial Cells: Implications for Excipients Safety.

Amphipathic, nonionic, surfactants are widely used in pharmaceutical, food, and agricultural industry to enhance product features; as pharmaceutical excipients, they are also aimed at increasing cell membrane permeability and consequently improving oral drugs absorption. Here, we report on the concentration- and time-dependent succession of events occurring throughout and subsequent exposure of Caco-2 epithelium to a "typical" nonionic surfactant (Kolliphor HS15) to provide a molecular explanation for nonionic surfactant cytotoxicity. The study shows that the conditions of surfactant exposure, which increase plasma membrane fluidity and permeability, produced rapid (within 5 min) redox and mitochondrial effects. Apoptosis was triggered early during exposure (within 10 min) and relied upon an initial mitochondrial membrane hyperpolarization (5-10 min) as a crucial step, leading to its subsequent depolarization and caspase-3/7 activation (60 min). The apoptotic pathway appears to be triggered prior to substantial surfactant-induced membrane damage (observed ≥60 min). We hence propose that the cellular response to the model nonionic surfactant is triggered via surfactant-induced increase in plasma membrane fluidity, a phenomenon akin to the stress response to membrane fluidization induced by heat shock, and consequent apoptosis. Therefore, the fluidization effect that confers surfactants the ability to enhance drug permeability may also be intrinsically linked to the propagation of their cytotoxicity. The reported observations have important implications for the safety of a multitude of nonionic surfactants used in drug delivery formulations and to other permeability enhancing compounds with similar plasma membrane fluidizing mechanisms.

Niosomes decorated with dual ligands targeting brain endothelial transporters increase cargo penetration across the blood-brain barrier

Nanoparticles targeting transporters of the blood-brain barrier (BBB) are promising candidates to increase the brain penetration of biopharmacons. Solute carriers (SLC) are expressed at high levels in brain endothelial cells and show a specific pattern at the BBB. The aim of our study was to test glutathione and ligands of SLC transporters as single or dual BBB targeting molecules for nanovesicles. High mRNA expression levels for hexose and neutral amino acid transporting SLCs were found in isolated rat brain microvessels and our rat primary cell based co-culture BBB model. Niosomes were derivatized with glutathione and SLC ligands glucopyranose and alanine. Serum albumin complexed with Evans blue (67 kDa), which has a very low BBB penetration, was selected as a cargo. The presence of targeting ligands on niosomes, especially dual labeling, increased the uptake of the cargo molecule in cultured brain endothelial cells. This cellular uptake was temperature dependent and could be decreased with a metabolic inhibitor and endocytosis blockers filipin and cytochalasin D. Making the negative surface charge of brain endothelial cells more positive with a cationic lipid or digesting the glycocalyx with neuraminidase elevated the uptake of the cargo after treatment with targeted nanocarriers. Treatment with niosomes increased plasma membrane fluidity, suggesting the fusion of nanovesicles with endothelial cell membranes. Targeting ligands elevated the permeability of the cargo across the BBB in the culture model and in mice, and dual-ligand decoration of niosomes was more effective than single ligand labeling. Our data indicate that dual labeling with ligands of multiple SLC transporters can potentially be exploited for BBB targeting of nanoparticles.

Concurrently suppressing multidrug resistance and metastasis of breast cancer by co-delivery of paclitaxel and honokiol with pH-sensitive polymeric micelles.

Multidrug resistance (MDR) and metastasis are considered to be two of the major barriers for successful chemotherapy. The combination of a chemotherapeutic drug with a modulator has emerged as a promising strategy for efficiently treating MDR cancer and preventing tumor metastasis. Herein, a dual drug (paclitaxel and honokiol)-loaded pH-sensitive polymeric micelle system based on PEOz-PLA was successfully fabricated to ensure that tumor MDR and metastasis could be concurrently suppressed, therefore achieving distinguishing endo/lysosomal pH from physiological pH by accelerating drug release and then enhancing the cytotoxicity of paclitaxel to drug-resistant tumor cells MCF-7/ADR by increasing cellular uptake of paclitaxel, preventing in vitro invasion and migration for MDA-MB-231 cells and in vivo metastasis in nude mice. Further, the mechanism of MDR reversal by dual drug-loaded PEOz-PLA micelles was elucidated to be down-regulation of P-gp expression and increase of plasma membrane fluidity of MCF-7/ADR cells. The present findings strengthen the usefulness of co-delivery of PTX and HNK by pH-responsive polymeric micelles for suppression of tumor MDR and metastasis.

Comparison of the effects of major fatty acids present in the Mediterranean diet (oleic acid, docosahexaenoic acid) and in hydrogenated oils (elaidic acid) on 7-ketocholesterol-induced oxiapoptophagy in microglial BV-2 cells

Increased levels of 7-ketocholesterol (7KC), which results mainly from cholesterol auto-oxidation, are often found in the plasma and/or cerebrospinal fluid of patients with neurodegenerative diseases and might contribute to activation of microglial cells involved in neurodegeneration. As major cellular dysfunctions are induced by 7KC, it is important to identify molecules able to impair its side effects. Since consumption of olive and argan oils, and fish is important in the Mediterranean diet, the aim of the study was to determine the ability of oleic acid (OA), a major compound of olive and argan oil, and docosahexaenoic acid (DHA) present in fatty fishes, such as sardines, to attenuate 7KC-induced cytotoxic effects. Since elaidic acid (EA), the trans isomer of OA, can be found in hydrogenated cooking oils and fried foods, its effects on 7KC-induced cytotoxicity were also determined. In murine microglial BV-2 cells, 7KC induces cell growth inhibition, mitochondrial dysfunctions, reactive oxygen species overproduction and lipid peroxidation, increased plasma membrane permeability and fluidity, nuclei condensation and/or fragmentation and caspase-3 activation, which are apoptotic characteristics, and an increased LC3-II/LC3-I ratio, which is a criterion of autophagy. 7KC is therefore a potent inducer of oxiapoptophagy (OXIdation+APOPTOsis+autoPHAGY) on BV-2 cells. OA and EA, but not DHA, also favor the accumulation of lipid droplets revealed with Masson’s trichrome, Oil Red O, and Nile Red staining. The cytotoxicity of 7KC was strongly attenuated by OA and DHA. Protective effects were also observed with EA. However, 7KC-induced caspase-3 activation was less attenuated with EA. Different effects of OA and EA on autophagy were also observed. In addition, EA (but not OA) increased plasma membrane fluidity, and only OA (but not EA) was able to prevent the 7KC-induced increase in plasma membrane fluidity. Thus, in BV-2 microglial cells, the principal fatty acids of the Mediterranean diet (OA, DHA) were able to attenuate the major toxic effects of 7KC, thus reinforcing the interest of natural compounds present in the Mediterranean diet to prevent the development of neurodegenerative diseases.

Plasma membrane order and fluidity are diversely triggered by elicitors of plant defence.

Although plants are exposed to a great number of pathogens, they usually defend themselves by triggering mechanisms able to limit disease development. Alongside signalling events common to most such incompatible interactions, modifications of plasma membrane (PM) physical properties could be new players in the cell transduction cascade. Different pairs of elicitors (cryptogein, oligogalacturonides, and flagellin) and plant cells (tobacco and Arabidopsis) were used to address the issue of possible modifications of plant PM biophysical properties induced by elicitors and their links to other events of the defence signalling cascade. We observed an increase of PM order whatever the elicitor/plant cell pair used, provided that a signalling cascade was induced. Such membrane modification is dependent on the NADPH oxidase-mediated reactive oxygen species production. Moreover, cryptogein, which is the sole elicitor able to trap sterols, is also the only one able to trigger an increase in PM fluidity. The use of cryptogein variants with altered sterol-binding properties confirms the strong correlation between sterol removal from the PM and PM fluidity enhancement. These results propose PM dynamics as a player in early signalling processes triggered by elicitors of plant defence.

Optimizing the freezing rate for ovine semen cryopreservation: Phospholipid profiles and functions of the plasma membrane and quality and fertilization of spermatozoa

The objective of this study was to investigate the effects of freezing rate on plasma membrane (PM) phospholipids and the function and fertilization of ram spermatozoa during freezing. Semen was obtained from 10 Merino rams using an artificial vagina, diluted with glucose-egg yolk buffer, and frozen in a programmable freezer at rates of −1, −20, −40, −60, and −80°C/min. After thawing, we examined PM phospholipid content and distribution, fluidity, permeability, potential, spermatozoa quality, and in vivo and in vitro fertilization rates. The results showed that phospholipid, phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) contents of the PM decreased significantly (P<0.05), whereas diphosphatidylglycerol (DPG) content and the cholesterol: phospholipid (CH: PL) ratio increased significantly compared to those of fresh semen after freezing (P<0.05). PE, phosphatidylinositol (PI), and DPG were transferred to the outer leaflet, whereas PS and PG were transferred to the inner leaflet after freezing. Spermatozoa cryopreserved at −40°C/min resulted in distributions of PS, PE, and DPG, as well as increased PM fluidity, permeability, and potential that was greater than those of the other freezing-rate groups (P<0.05). No differences in vitality, PM and acrosome integrity, in vitro fertilization hatching rate, or artificial insemination conception rate were observed in any of the freezing-rate groups (P>0.05), whereas cleavage rate (69.67±5.11%) at −40°C/min increased significantly (P<0.05). Therefore, the optimal freezing rate to cryopreserve ram spermatozoa based on cryoinjury of phospholipids, membrane functions, and fertilization rate was −40°C/min.

Abstract B2-10: Systems biology reveals role of cholesterol homeostasis in breast cancer metastasis

Abstract New therapeutic targets that can inhibit cancer metastasis are desperately needed. To address this issue, we have leveraged a computational approach and performed an unbiased screen for therapies that can inhibit an epithelial-to-mesenchymal transition (EMT) gene expression profile, a phenotype that is believed to promote metastases. This yielded predictions of drugs that can both promote and inhibit an EMT transcriptional profile. Following up on these predictions, we confirmed experimentally that these drugs can induce or repress an EMT in a range of breast cancer cell lines. Furthermore, we discovered that they do so by altering the fluidity of the plasma membrane. An increase in plasma membrane fluidity is necessary and sufficient to induce and maintain an EMT state, increased motility, and increased mammosphere forming ability. Finally, we found that human cancers can regulate their membrane fluidity through cholesterol homeostasis pathways. Human breast tumors with altered cholesterol profiles have a significantly shorter time to metastasis. These data demonstrate that an unbiased systems approach can be used to discover novel pathways that underlie metastasis. Our multidisciplinary approach combining computational, cell biology, and mouse models reveal that plasma membrane fluidity is a cellular attribute that is leveraged by human tumors to increase metastatic capacity, and that it can, in principle, be targeted by therapeutics to prevent metastasis. Citation Format: Weina Zhao, Prijic Sara, Bettina Urban, Michael Tisza, Sendurai A. Mani, Jeffrey T. Chang. Systems biology reveals role of cholesterol homeostasis in breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B2-10.

Sphingolipids modulate the epithelial-mesenchymal transition in cancer.

Sphingolipids modulate the epithelial-mesenchymal transition in cancer.

Methyl-β-Cyclodextrin Improves Sperm Capacitation Status Assessed by Flow Cytometry Analysis and Zona Pellucida-Binding Ability of Frozen/Thawed Bovine Spermatozoa.

Mammalian sperm undergo a series of biochemical transformations in the female reproductive tract that are collectively known as capacitation. Cyclodextrins added to the sperm culture medium have been described to induce in vitro sperm capacitation, enabling its use in protein-free media. However, the additive capacitating effect of methyl-β-cyclodextrin (MβCD) in the medium containing bovine serum albumin (BSA) is unknown in the bovine species. In this study, we evaluated the effects of incubating frozen-thawed bovine spermatozoa in a BSA-containing medium supplemented with MβCD on different sperm quality and functional parameters. Sperm viability decreased with the addition of MβCD in a dose-dependent manner (p < 0.05), and DNA damage could be observed but only with the highest concentration of MβCD. However, pre-incubation of spermatozoa in MβCD-supplemented medium improved the capacitation status as assessed by the increase in plasma membrane fluidity, intracellular calcium concentration, induced acrosome reactivity and zona pellucida (ZP)-binding ability (p < 0.05). Thus, we conclude that MβCD supplementation is able to enhance the capacitation status of frozen-thawed bovine spermatozoa cultured in capacitation medium containing BSA and could result in a valid strategy for its application on artificial reproductive technologies such as in vitro fertilization or intracytoplasmic sperm injection.

Chemotherapy with ceramide in TNBC.

Metastasis remains the major cause of cancer-related mortality, and currently there is a lack of therapies to eliminate this risk. Therefore, it is of crucial interest to better understand this multi-step cellular process and find the best markers to identify patients who will develop early and distant relapse. This latter objective may allow clinicians to adapt their treatments and propose combination of therapeutic drugs to patients who are at the highest risk of metastasis. CD95L belongs to the Tumor Necrosis Factor (TNF) superfamily and binds CD95 (also known as Fas). In contrast to its ubiquitously expressed receptor, CD95L exhibits a restricted expression pattern, being mainly found at the surface of activated T lymphocytes and natural killer (NK) cells where it contributes to the elimination of transformed and infected cells. Metalloproteases can cleave CD95L leading to its release in the bloodstream. While transmembrane CD95L (m-CD95L) is a potent apoptotic inducer that participates in immune surveillance and tolerance, metalloprotease-cleaved CD95L (cl-CD95L) fails to trigger apoptosis, but implements non-apoptotic signaling pathways (PI3K, K-Ras, NFkB, MAPK) promoting cell survival, proliferation and migration [1]. By doing so, CD95 behaves as an oncogene. We recently established that high concentrations of serum CD95L are present in triple negative breast cancers (TNBC). Among breast cancers, TNBCs are characterized by lack of estrogen and progesterone receptor expressions and absence of HER2 overexpression. Unlike others breast cancer categories, TNBC women do not benefit from targeted therapies and thereby, these patients relapse earlier than other breast cancer patients. Importantly, high amounts of cl-CD95L in TNBC women are associated with poor prognoses and administration of cl-CD95L in mice promotes the metastatic dissemination of TNBC cells [2]. Epithelial to mesenchymal transition (EMT) is a cellular reprogramming that plays pivotal role in embryonic development and that can be partly phenocopied in carcinogenesis, giving rise to aggressive tumors with increased metastatic capacity. EMT reprograms the apoptotic machinery in cancer cells to allow them to resist CD95-mediated cell death [3]. But not only, we recently demonstrated that EMT also enhances plasma membrane fluidity and thereby, promotes cell migration of breast cancer cells [4]. Sphingolipids (SLs) are sphingoid base-containing lipids, which are membrane components enriched in membrane microdomains, being concentrated into the outer leaflet of the plasma membrane and modulating its biochemical and biophysical properties such as membrane fluidity. EMT is associated with changes in SL metabolism, and some SL metabolites, such as gangliosides (i.e., sialic acid-containing glycosphingolipids), can affect this cellular reprograming [5]. An integrative analysis of the gene expression in NCI-60 tumor cell lines revealed that the expression level of a ceramide synthase, namely ceramide synthase-6 (CerS6) decreases during EMT. CerS6 expression level mainly regulates the amount of C16:0-ceramide in cancer cells. Interestingly, whereas reducing the amounts of C16:0- ceramide in TNBC cells by down-regulating CerS6 expression level, increased plasma membrane fluidity and promoted cell motility, exogenous addition of non-cytotoxic doses of C16:0-ceramide or over-expression of CerS6 in TNBC cells led to a stiffening of plasma membrane, abrogating both basal and cl-CD95L-mediated cell migration [4]. These findings led us to postulate that administration of low doses (that may reduce side effects for the patients) of C16:0-ceramide in combination with chemotherapy in TNBC women may apply the brake on metastatic dissemination. Like CD95L, tumor-necrosis-factor related apoptosis-inducing ligand (TRAIL) belongs to the TNF family. In line with CD95's pro-metastatic potential, DR5 (TRAIL-R2), one of the two agonistic TRAIL receptors, can promote breast cancer skeletal metastasis in a mouse xenograft model [6]. Earlier evidence already pointed to TRAIL promoting migration in TRAIL-resistant colon carcinomas [7]. More recently, it has been reported that DR5 displays pro-metastatic activity in KRAS derived non-small-cell lung cancers and pancreatic ductal adenocarcinoma (PDAC), and high expression levels of DR5 are correlated with increased invasion and reduced metastasis-free survival in KRAS mutated patients suffering from PDAC or colorectal cancer, respectively [8]. Although, regulation of SL membrane composition induced by TRAIL, is mostly ascribed to its pro-apoptotic potential, changes in SL content are also likely to contribute to regulation of DR5 pro-metastatic potential. This growing body of evidence put forward that like TNF-R, CD95 and DR5 might correspond to pro-inflammatory receptors modulating the quantity and composition of SLs in cancer cells to affect the biophysical properties of their plasma membrane. These observations raise now the question of how the so-called “death receptors” can affect the SL metabolism to enhance cancer cell migration. Identification of the molecular mechanism linking these receptors to SL metabolism is likely to reveal new therapeutic options and targets to prevent tumor dissemination in cancers.

Abstract 4984: EMT and breast cancer metastasis driven by plasma membrane fluidity

Abstract New therapeutic targets that can inhibit cancer metastasis are desperately needed. To address this issue, we have leveraged a computational approach and performed an unbiased screen for therapies that can inhibit an epithelial-to-mesenchymal transition (EMT) gene expression profile, a phenotype that is believed to promote metastases. This yielded predictions of drugs that can both promote and inhibit an EMT transcriptional profile. Following up on these predictions, we confirmed that these drugs can induce or repress an EMT in a range of breast cancer cell lines. Furthermore, we discovered that they do so by altering the fluidity of the plasma membrane. An increase in plasma membrane fluidity is necessary and sufficient to induce and maintain an EMT state, increased motility, and increased mammosphere forming ability. Therapies that decrease fluidity can inhibit metastasis in xenograft mouse models. Finally, we found that human cancers can regulate their membrane fluidity through cholesterol homeostasis pathways. 41% of human breast cancers have alterations in cholesterol regulatory pathways, and are associated with a 9 year loss in time to distant metastasis. These data demonstrate that plasma membrane fluidity is a cellular attribute that is leveraged by human tumors to increase metastatic capacity, and that it can, in principle, be targeted by therapeutics to prevent metastasis. Citation Format: Jeffrey T. Chang. EMT and breast cancer metastasis driven by plasma membrane fluidity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4984. doi:10.1158/1538-7445.AM2015-4984