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Lipid rafts, cholesterol and apoptosis in cancer and neurodegenerative diseases Faustino Mollinedo and Consuelo Gajate

Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer,

CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain

Running title: Cholesterol-rich lipid rafts in cancer and neurodegenerative diseases

Corresponding authors:

Faustino Mollinedo and Consuelo Gajate, Instituto de Biología Molecular y Celular del

Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus

Miguel de Unamuno, E-37007 Salamanca, Spain. Phone: (+34) 923-294806, Fax: (+34)

923-294795, E-mail: fmollin@usal.es. E-mail: cgajate@usal.es.

ABSTRACT

The dynamic nature of membrane along with an uneven distribution of lipids leads to the

formation of specialized membrane domains where proteins can selectively be included or

excluded. In this regard, the dynamic and preferential clustering and packing of

sphingolipids and cholesterol into moving platforms, named as lipid rafts, form membrane

domains that act as scaffolds for the attachment of specific proteins and for the proper

functioning of a number of signaling cascades. Lipid rafts harbor several signaling routes

that promote cell survival and proliferation, and thereby they could play a role in the

development of cancer. On the other hand, evidences in the last decade have shown that

lipid rafts can also serve as organizing centers for the assembly of apoptotic and signaling

molecules involved in cell death, thus opening a new avenue in cancer therapy. Recruitment

of death receptors and downstream apoptotic signaling molecules in aggregated lipid rafts

has led to the emerging concept of a plasma membrane platform designated as “cluster of

apoptotic signaling molecule-enriched rafts” (CASMER) that may play a role in the

regulation of apoptosis. In addition, lipid rafts also play a major role in neurotoxicity,

including the production and aggregation of amyloid-β peptide (Aβ) to form neurotoxic Aβ

oligomers in the brain, which are widely believed to drive Alzheimer’s disease pathology.

Alterations in cholesterol have been detected in cell models for cancer and

neurodegenerative diseases, which might lead to changes in cholesterol-rich rafts and

thereby in the regulation of cell death or survival. However, it is currently unclear what the

potential mechanisms underlying cholesterol perturbations are. Growing evidence suggests

that compartmentalization of macrocomplexes and signaling routes in lipid rafts has a

crucial role in the regulation of cell fate, being of major importance in different pathologies,

including cancer and neurodegenerative diseases. Despite the underlying mechanisms and

functional impact of protein compartmentalization are still not well understood, the

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modulation of lipid rafts opens new approaches in the treatment of these diseases for which

current available therapies are not satisfactory.

INTRODUCTION

The role of apoptosis in normal physiology is as significant as that of its counterpart,

mitosis, in the regulation of cell populations. In adulthood, about 10 billion cells die every

day simply to keep balance with the numbers of new cells arising from the body’s stem cell

populations (1). This normal homeostasis is tightly regulated through apoptosis.

Abnormalities in cell death regulation can be a significant component of serious diseases,

such as cancer, associated with deficient apoptosis, and neurodegenerative disorders,

associated with excessive apoptosis. In this regard, emerging evidence suggests that insight

in how cells handle cholesterol seems to be of major importance for keeping the number of

cell populations constant within narrow limits, and breakdown of cholesterol homeostasis

may cause several disease states (2). Despite cholesterol accumulation in tumors was first

reported in the early 20th

century (3-5), its role in cancer development remains to be

elucidated. Subsequent reports also supported a higher cholesterol content in tumor cells

than in their normal counterparts, and there is now increasing evidence to suggest a link

between cholesterol accumulation and the risk of certain malignancies (6-10). Several

epidemiologic studies have described a reduced incidence of certain cancers in patients

taking 3-hydroxy-3-methyl-glutaryl CoA (HMG-CoA) reductase inhibitors (“statins”) for

cardiovascular indications (11-16), which inhibit the rate-limiting step of conversion of

HMG-CoA to mevalonate in cholesterol biosynthesis.

In addition, evidence has accumulated for the last 2 decades in support of the

hypothesis that elevated cholesterol levels increase the risk of developing Alzheimer’s

disease (AD) (17,18). The brain is particularly enriched in cholesterol, which is essential for

neuronal development and survival, maintenance of membrane integrity in neurons, synapse

maturation, and optimal synaptic activity. About 23-25 percent of the body's total

cholesterol level is found in the central nervous system (19), and alterations in brain

cholesterol homeostasis are linked to neurodegeneration. Recent findings suggest that

alterations in cholesterol homeostasis are associated with several neurodegenerative

disorders, including Huntington’s disease and AD (20-23).

The advent of the concept of lipid rafts in 1997 (24), as membrane domains enriched

in cholesterol and sphingolipids, has changed dramatically our vision of the role of

cholesterol in cell function. Lipid rafts are membrane microdomains of reduced fluidity

consisting of dynamic assemblies of cholesterol and sphingolipids (25,26). The presence of

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saturated hydrocarbon chains in sphingolipids allows for cholesterol to be tightly

intercalated, leading to the presence of distinct liquid-ordered phases, i.e. membrane rafts,

dispersed in the liquid-disordered matrix, and thereby more fluid lipid bilayer. Thus, lipid

rafts are more ordered and tightly packed membrane domains than the surrounding bilayer

(27,28). The hydrophobic chains of lipids in the rafts are more saturated and tightly packed

than the surrounding bilayer. Cholesterol is the dynamic glue that holds the raft together and

influences lipid-protein interactions by increasing the thickness of the lipid bilayer. One of

the most important features of lipid rafts is that they can include or exclude proteins. On

these grounds, these dynamic specialized membrane raft microdomains of reduced fluidity

may serve as foci for recruitment of distinct molecules at the plasma membrane (25,26,29),

thus playing a major role in the compartmentalization of cellular processes, including signal

transduction and membrane protein trafficking (25,30).

LIPID RAFTS IN CANCER DEVELOPMENT

Lipid rafts form platforms for individual receptors activated by ligand binding. This

activation could lead to the recruitment in the membrane raft of appropriate signaling

molecules, and signal transmission could be protected from non-raft enzymes, such as

membrane phosphatases, that otherwise could affect the signaling process. Thus, raft binding

recruits proteins to a new microenvironment, where the phosphorylation state of proteins can

be modified by local kinases and phosphatases, resulting in downstream signaling (25). On

these grounds, recruitment, retention and exclusion of certain proteins in lipid rafts leads to

identify these membrane domains as privileged sites where the interaction between discrete

subsets of signaling molecules is highly favored, thereby serving as platforms for signal

transduction (25). Thus, spatial compartmentalization of signaling pathway components

generally defines the specificity and enhances the efficiency of signal transduction. A wide

number of signal transduction process have been reported to occur in lipid rafts, including

those related to immune system (T-cell receptor, B-cell receptor, FcεRI receptor) and cell

survival and proliferation (insulin receptor, H-Ras, EGF receptor) (25).

Interestingly, cholesterol-rich rafts mediate Akt signaling in cancer cells (5,10).

Activated phosphatidylinositol 3-kinase (PI3K) and its downstream target Akt/PKB are

important signaling molecules and key survival factors involved in the control of cell

proliferation, apoptosis and oncogenesis. Aberrant activation of PI3K-Akt pathway may

contribute to the development and invasiveness of cancer cells (31,32). Thus, because PI3K-

Akt pathway is compartmentalized within plasma membrane raft domains (33), as well as

some additional survival signaling pathways, these membrane domains may play a role in

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cancer progression (34). Furthermore, lipid rafts play a crucial role in the localization and

functionality of CD44, which regulates cancer cell adhesion and migration (35).

Interestingy, treatment of human glioma cells with the lipid raft disrupting agent methyl-β-

cyclodextrin led to an increase in CD44 (35). Thus, low cholesterol triggers raft-dependent

CD44 shedding and suppresses tumor cells migration (35). The cholesterol-lowering

medication symvastatin has also been reported to enhance CD44 shedding, and to block the

stimulation of glioma cell migration by hyaluronan oligosaccharides or epidermal growth

factor (36-39). Thus, these results suggest that lowering cholesterol levels may disturb the

regulated CD44 membrane localization in rafts that is necessary for enhanced cancer cell

adhesion and migration (39). Unlike CD44, which was present in lipid rafts, its processing

enzyme, a disintegrin and metalloproteinase 10 (ADAM10), was largely in non-raft fractions

(35). Displacement of CD44 from lipid raft to non-raft membrane domains, where ADAM10

is located, might make CD44 accessible to ADAM10 and thereby to CD44 shedding (39).

An increasing number of proteins involved in the development of several malignant cancers

are being detected to be associated with lipid rafts (40), such as the type 1 transmembrane

glycoprotein mucin 1 (MUC1) (41) and urokinase plasminogen activator receptor (uPAR)

(42), suggesting major role of lipid rafts in tumor progression. In this regard, the cholesterol

content in tumor cells has been reported to be higher than in normal cells (6-9,43). Likewise,

cancer cells have been suggested to display higher levels of cholesterol-rich lipid rafts than

their normal counterparts (44).

LIPID RAFTS IN CANCER CHEMOTHERAPY

While investigating the mechanism of action of the antitumor ether phospholipid edelfosine

1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, ET-18-OCH3), the prototype of a

heterogenous family of compounds collectively known as synthetic alkyl-lysophospholipid

analogues (ALPs) or antitumor lipids (ATLs) (45,46), we found in 2000 that the induction of

apoptosis triggered by this drug in leukemic cells was dependent on the death receptor

Fas/CD95, which resulted aggregated at one pole of the cell to form caps on the cell surface

(47). Soon afterwards in 2001, we found out that edelfosine prompted translocation and co-

clustering of the death receptor Fas/CD95 into membrane rafts, thus uncovering a new

molecular process in the regulation of apoptosis through membrane rafts (48). This new

mechanism of action involved for the first time membrane rafts in Fas/CD95-mediated

apoptosis and cancer chemotherapy (48). Confocal microscopy analyses (Figure 1) and

isolation of membrane rafts through sucrose gradient centrifugation showed co-aggregation

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of Fas/CD95 and lipid rafts after incubation of Jurkat T-cell leukemia cells with edelfosine.

Raft disruption, following treatment with the cholesterol-depleting agent methyl- -

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lfosine-induced Fas/CD95 clustering and apoptosis (48). These results showed that lipid

rafts could not only harbor molecules related to survival and proliferating signaling, as

mentioned above, but also cell death signaling molecules, such as death receptor Fas/CD95.

Subsequent evidences showed that that Fas/CD95 was also translocated into lipid

rafts following activation with its natural ligand FasL/CD95L (49,50). On these grounds,

edelfosine mimicked to some extent the action of the natural ligand FasL/CD95L on

promoting death receptor clustering in lipid rafts. However, translocation of Fas/CD95 into

membrane rafts following edelfosine treatment was independent of receptor interaction with

FasL/CD95L (47,48). Furthermore, Fas/CD95 was triggered intracellularly by edelfosine, so

the drug was acting from within the cell independently of FasL/CD95L (51). Thus, these

data indicate that Fas/CD95 aggregation in rafts and its ensuing activation can be modulated

pharmacologically.

Additional studies have shown that edelfosine not only induced recruitment of the

death receptor Fas/CD95 in lipid rafts, but also promoted translocation of downstream

signaling molecules into rafts. Fas-associated death domain protein (FADD) and procaspase-

8, which together with Fas/CD95 form the death inducing signaling complex (DISC),

required to mount an apoptosis signal, were also recruited into membrane rafts upon

edelfosine treatment in a number of hematopoietic cancer cells (51-53). Formation of DISC

was visualized upon edelfosine treatment by electron microscopy (Figure 2) and co-

immunoprecipitation assays (52,53). A number of additional antitumor drugs have been

recently reported to promote the recruitment of the three major constituents of DISC, namely

Fas/CD95, FADD and procaspase-8, into lipid rafts, including perifosine (52), cis-platin

(54), resveratrol (55,56), aplidin (57), rituximab (58), and avicin D (59).

Aggregation of death receptors in a rather small area of the cell, such as lipid raft

membrane domains, following antitumor chemotherapy, would allow a potent synergy with

death receptor ligands to achieve cell death. On these grounds, a number of antitumor drugs,

including resveratrol (56,60), aplidin (57), edelfosine (52) and perifosine (52) have been

reported to recruit death receptors Fas/CD95, tumor necrosis factor receptor 1 (TNFR1) and

TNF-related apoptosis-inducing ligand (TRAIL) receptors, named death receptor (DR) 4 and

DR5, into lipid rafts, and this protein redistribution sensitized the cells to death receptor

stimulation by their cognate ligands or agonistic cytotoxic antibodies (52,60). The above DISC

apoptotic complex can also be formed with the death receptors DR4 and DR5(61-63), and these

TRAIL receptor-mediated DISCs are also suggested to be recruited into rafts following

treatment with a number of antitumor drugs (52,56,60).

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Interestingly, some anticancer drugs, including edelfosine (51,64), perifosine (65) and

aplidin (57), have been reported to accumulate in lipid rafts and to reorganize these

membrane domains, affecting their protein composition. Thus, these data indicate that lipid

rafts can be targeted by chemical compounds, leading to a new way to regulate cell fate,

either promoting apoptosis or survival.

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APOPTOTIC SIGNALING MOLECULES IN LIPID RAFTS

In addition to DISC constituents, an increasing number of proteins involved in cell death

regulation are being reported to be accumulated in membrane rafts upon treatment of cancer

cells with antitumor drugs. In this regard, downstream apoptotic signaling molecules,

including procaspase-10, c-Jun N-terminal kinase (JNK), and BH3-interacting domain death

agonist (Bid) have been found to be translocated into membrane rafts of cancer cells

following edelfosine treatment (51,52,66,67). Persistent JNK activation is associated with

apoptosis (68,69), and Bid acts as a connector between Fas/CD95-mediated extrinsic

signaling and the mitochondrial-dependent intrinsic pathway of apoptosis (70). The

antitumor drugs aplidin (57) and resveratrol (56) also induce recruitment of JNK and Bid in

membrane rafts in human leukemic cells. This may explain the dependence of edelfosine-,

aplidin- and resveratrol-mediated apoptosis on both JNK and mitochondrial signaling

(56,69,71,72). Redistribution of death receptors and downstream signaling molecules into

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lipid rafts does not require protein synthesis, and therefore it is achieved from the pre-

existing protein pool (51).

THE CONCEPT OF CASMER IN THE REGULATION OF APOPTOSIS

Recruitment of death receptors and downstream signaling molecules in lipid rafts is

expected to facilitate and potentiate protein-protein interactions and cross-talk between

different signaling pathways, which could eventually lead to the triggering of cell death

signals. The outstanding recruitment of death receptors, together with downstream apoptotic

signaling molecules, in aggregated rafts has led us to coin the word CASMER as an

acronym of “cluster of apoptotic signaling molecule-enriched rafts” (57,67,73,74).

CASMER refers to the recruitment of death receptors together with downstream apoptotic

signaling molecules in aggregated rafts (73,74). CASMER represents a novel raft-based

supramolecular entity, acting as death-promoting platforms where death receptors and

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ng molecules are brought together (Figure 3). Thus, CASMER formation would facilitate

protein-protein interactions and the transmission of apoptotic signals. The efficiency in

promoting CASMER formation, as well as CASMER protein composition, would depend on

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the cell phenotype and the triggering stimulus (73,74). A basic protein composition of

CASMER would include the recruitment of death receptors in aggregated rafts (73,74), but

CASMERs could increase in complexity by recruiting additional downstream signaling

molecules in lipid rafts, including DISC components FADD and procaspase-8, Bid, and JNK

(52,53,73-75). The higher number of proteins recruited in CASMERs, the more efficient the

apoptosis response launched. CASMER formation seems to play an important role in cross-

talk processes occurring during apoptosis. In this regard, the recruitment of Bid in

membrane rafts highlights a major role of rafts as a putative connector between extrinsic and

intrinsic signaling pathways in apoptosis (51,52,56,57,67).

LIPID RAFTS IN NEURODEGENERATIVE DISEASES

A growing amount of evidence suggests a mechanistic link between cholesterol metabolism

in the brain and the formation of amyloid plaques in AD development (76). In addition,

recent evidence suggests that ganglioside GM1, another component of rafts, facilitates the

binding and accumulation of Aβ oligomers at lipid rafts (77,78). Lipid rafts play a number of

critical roles in AD, namely promoting the generation of the amyloid-β peptide (Aβ),

facilitating its aggregation upon neuronal membranes to form toxic oligomers and hosting

neuronal receptors through which AD-related neurotoxicity of the Aβ oligomers is

transduced (79). Neuronal sensitivity to Aβ–induced toxicity has been found to be dependent

upon Aβ binding to the cell membrane (80). Soluble Aβ dimers accumulate rapidly, and

have been found at elevated levels, in lipid rafts from human and transgenic mouse model

mouse AD brains (81), as well as in presynaptic terminals in cholesterol- and ganglioside

GM1-rich lipid rafts from AD cortex (82). Cholesterol modulates the interaction of the Aβ

peptide with lipid bilayers (83). Aβ oligomers isolated from AD patients associate with lipid

rafts in a cholesterol-dependent way, and cholesterol depletion decreases Aβ aggregation

(84). Additional studies have revealed that an increase in the level of cholesterol in human

neuroblastoma cells reduces Aβ oligomer binding (85,86), suggesting that a fluctuation in

cholesterol levels may alter the physical properties of lipid rafts thereby modulating Aβ

oligomer binding (86). Cholesterol and statins clearly modulate β-amyloid precursor protein

processing in cell culture and animal models (76). In addition, Aβ oligomer binding induces

the clustering of neuronal receptors into aberrant pathogenic signaling platforms (87), and

thereby lipid rafts may represent platforms for Aβ oligomer-mediated neurotoxicity (79).

Lipid rafts can also play a key role in a range of neurodegenerative disease, in addition to

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AD, including Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and

prion disease (88).

Platelet-activating factor (1-O-hexadecyl-2-acetyl-sn-glycerophosphocholine, PAF)

has been implicated in the neuronal damage that accompanies ischemia, prion disease and

AD. PAF is elevated in the brains of AD patients and in human neurons exposed to Aβ (89),

and chronic PAF accumulation leads to an endoplasmic reticulum stress-mediated neuronal

death (89). Thus, high concentrations of PAF are thought to contribute to neuronal damage.

On the other hand, studies using PAF antagonists have indicated that PAF is involved in

neuronal loss in AD (90). The effects of PAF are mediated via a specific receptor (91),

which is located in neuronal lipid rafts (92). Treatment of neurons with cholesterol synthesis

inhibitors, including simvastatin and squalestatin, disrupted cholesterol-rich lipid rafts, and

protected neurons against PAF through a desensitisation of neurons to PAF, likely as a result

of a displacement of PAF receptors to non-raft membranes (92).

CONCLUSIONS

Recent advances in our understanding of the organization, composition and function of lipid

rafts in cancer cells have led to the conclusion that these membrane domains act as platforms

for the recruitment of signaling processes that regulate cell fate, promoting either survival or

cell death. Lipid rafts harbor a wide number of signaling cascades involved in survival and

cell proliferation, thus potentiating cancer development. In this regard, elevated levels of

cholesterol and lipid rafts have been reported in cancer cells as compared to their normal

counterparts. However, in the last years increasing evidence has been accumulated for an

important role of lipid rafts in cell death, acting as scaffolds for death signaling pathways

and death-promoting processes. The finding that some drugs can accumulate in lipid rafts,

modulating their protein composition and function, opens exciting new avenues of research

to get insight into the regulation of cell death in important human pathologies, such as

cancer and neurodegenerative diseases. A better understanding of how lipid rafts modulate

cell fate, and how these membrane domains can be modulated, would lead to a significant

advance in the design of novel strategies in the treatment of cancer and neurodegenerative

diseases.

ACKNOWLEDGEMENTS

This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación

(SAF2008-02251, SAF2011-30518, and RD06/0020/1037 from Red Temática de

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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 Programme FP7-2007-2013 (grant HEALTH-F2-2011-256986), Junta de

Castilla y León (CSI052A11-2, GR15-Experimental Therapeutics and Translational

Oncology Program, Biomedicine Project 2009, and Biomedicine Project 2010-2011) to

F.M.; grants from the Fondo de Investigación Sanitaria and European Commission (FIS-

FEDER 06/0813, PS09/01915) to C.G. C.G. is supported by the Ramón y Cajal Program

from the Spanish Ministerio de Ciencia e Innovación.

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