Dissecting lipid raft facilitated cell signaling pathways in cancer

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Biochimica et Biophysica Acta 1785 (2008) 182 – 206 www.elsevier.com/locate/bbacan

Review

Dissecting lipid raft facilitated cell signaling pathways in cancer Samir Kumar Patra ⁎ Cancer Epigenetics Research, Kalyani (B–7/183), Nadia, West Bengal, India–741235 Received 4 October 2007; received in revised form 24 November 2007; accepted 29 November 2007 Available online 5 December 2007

Abstract Cancer is one of the most devastating disorders in our lives. Higher rate of proliferation than death of cells is one of the essential factors for development of cancer. The dynamicity of cell membrane plays some vital roles in cell survival and cell death, including protection, endocytosis, signaling, and increases in mechanical stability during cell division, as well as decrease of shear forces during separation of two cells after division, and cell separation from tissues for cancer metastasis. Within the membrane, there are specialized domains, known as lipid rafts. A raft can coordinate various signaling pathways. Recent data on the proteomics of lipid rafts/caveolae have highlighted the enigmatic role of various signaling proteins in cancer development. Analysis of these data of raft proteome from various tumors, cancer tissues, and cell lines cultured without and with therapeutic agents, as well as from model rafts revealed that there may be two subsets of raft assemblage in cell membrane. One subset of raft is enriched with cholesterol–sphingomyeline–ganglioside–cav-1/Src/EGFR (hereafter, “chol-raft”) that is involved in normal cell signaling, and when dysregulated promotes cell transformation and tumor progression; another subset of raft is enriched with ceramide–sphingomyeline–ganglioside–FAS/Ezrin (hereafter, “cer-raft”) that generally promotes apoptosis. In view of this, and to focus insight into the cancer cell physiology caused by the lipid rafts mediated signals and their receptors, and the downstream transmitters, either proliferative (for example, EGF and EGFR) or death-inducing (for example, FASL and FAS), and the precise roles of some therapeutic drugs and endogenous acid sphingomylenase in this scenario in in situ transformation of “chol-raft” into “cer-raft” are summarized and discussed in this contribution. © 2007 Elsevier B.V. All rights reserved. Keywords: Cancer; Catenin; Caveolin-1; CD44; Ceramide; EGFR; Cholesterol; E-cadherin; Ezrin; FAS/CD95; FASL; Focal adhesion kinase; H-ras; Integrin; Lipid rafts; Matrix metalloproteinases (MMPs); Proteomics; Signal transduction; Sphingomyelin; uPA; uPAR; MAP kinase

Contents 1. 2.

3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. The flora and fauna of lipid rafts. . . . . . . . . . . . . . . . . Lipid rafts/caveolae signaling and cancer . . . . . . . . . . . . . . . . 2.1. Raft and caveolin-1 signaling . . . . . . . . . . . . . . . . . . 2.2. Tumor suppression and metastasis promotion. The cav-1, CD44, Raft and epidermal growth factor receptor (EGFR) signaling . . . . . . Nuclear factor kappa B signaling . . . . . . . . . . . . . . . . . . . . Rafts in MAP kinase, Ras, and activator protein 1 (AP1) signaling . . .

. . . . . . . . . . . . . . . . . . . . . . . . E-cadherin . . . . . . . . . . . . . . . . . .

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Abbreviations: Acid sphingomyelinase, ASMase; Activator protein-1, AP-1; Caveolin-1, cav-1; Ceramide, Cer; Cholesterol, Chol; Extracellular matrix, ECM; E-cadherin, E-cad; Endoplasmic reticulum, ER; Epidermal growth factor, EGF; EGF receptor, EGFR; Extracellular signal-regulated kinase, ERK; FAS antigen, FAS; FAS associated death domain, FADD; FAS ligand, FASL, Death-inducing signaling complex, DISC; Focal adhesion kinase, FAK; Glycosyl phosphatidyl inositol, GPI; Insulin like growth factor, IGF; Matrix metalloproteinases, MMPs; Mitogen activated protein kinase, MAPK; MAP/ERK kinase 1/2, MEK1/2; Nuclear factor-kB, NF-kB; Phosphoinositide 3-kinase, PI3K; Plasma membranes, PM; Receptor tyrosine kinases, RTKs; Retinoic acid, RA; RA receptor, RAR; Sentinel lymph nodes, SLN; Sphingomyelin, SM; Thrombospondine 2, THBS2; Urokinase type plasminogen activator (uPA); uPA receptor (uPAR) ⁎ Corresponding author. Tel.: +91 9432060602; fax: +91 3325828460. E-mail address: [email protected]. 0304-419X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbcan.2007.11.002

S.K. Patra / Biochimica et Biophysica Acta 1785 (2008) 182–206

6. 7. 8. 9. 10. 11. 12. 13. 14.

Raft and insulin like growth factor mediated signaling . . . . . Extracellular matrix and raft signaling . . . . . . . . . . . . . Rafts in MMPs and uPAR signaling . . . . . . . . . . . . . . Rafts in integrin and focal adhesion kinase mediated signaling Raft signaling and apoptosis . . . . . . . . . . . . . . . . . . FAS and raft mediated FAS signaling . . . . . . . . . . . . . Rafts and ezrin signaling . . . . . . . . . . . . . . . . . . . . Acid sphingomyelinase and raft signaling . . . . . . . . . . . Discussion and perspectives . . . . . . . . . . . . . . . . . . 14.1. Tumour suppression and metastasis promotion duality. Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

1.1. The flora and fauna of lipid rafts

Maintenance of balance between cell proliferation and cell death is the main key of normal development. Cancer cells have higher rate of proliferation than death. Usually, deregulated cell cycle is the cause of such impairment. Deregulation of cell cycle is caused by aberrant signaling. The dynamicity of membranes and flip-flop flexibility of lipids within cell membrane play some vital roles in cells life and death. Plasma membrane gives a protection to cytoplasmic ingredients and organelles, it perform endocytosis and signaling, and increases the mechanical stability of cells during division, as well as the flexibility of lipids within the membrane causes decrease of shear forces during cell separation (for example, separation of individual cells from the host tumor during cancer metastasis). Within the membrane, there are specialized domains, known as lipid rafts. Many proteins of receptor tyrosine kinases (RTK) family members, including epidermal growth factor receptor (EGFR), and other proteins, including caveolin-1, CD44, uPAR, H-Ras, integrins and catenins have been implicated to various cellular functions, including stability and signaling. Some of these proteins precisely exhibit their function through lipid rafts, either structurally or functionally, or both, in immune signaling, angiogenesis, cell polarity and cancer progression. Function of proteins like, FAS and FASL virtually remain inert, which in turn facilitate tumor development by reduced rate of apoptosis. Also, impaired function of FAS and FASL results in tumor development, immune disorders and other diseases, including diabetes and Parkinson's disease. Investigations on the molecular mechanisms of cell transformation and development of various cancers, including breast, lung, prostate, gliomas and multiple sarcomas are given immense importance, since cancer is one of the major threats in our life. We have been working for years on molecular and epigenetic regulation of cancer, including lipid rafts, DNA methylation, and lipid rafts and cancer metastasis [1]. In this contribution, I shall discuss some important signaling events leading to cell transformation and cancer progression, which otherwise depend predominantly on lipid rafts. A handfull collective knowledge of lipid rafts and raftassisted signaling pathways would help us to choose strategies for prevention, cure and better management of cancers using natural compounds, synthetic inhibitors, radiation or other forms of therapies.

Lipid/membrane rafts are small (10–200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Caveolae, a subclass of rafts, are characterized by flask-like invaginations of the plasma membrane that are distinguished from bulk lipid rafts by the presence of caveolin-1 (cav-1). Hence, lipid rafts/caveolae are specialized molecular assemblages of sphingolipids and cholesterol, orchestrated by proteins and gangliosides that are known principally for their pivotal role in trans-cytosis, sorting of sphingolipids and cholesterol in the cell, and as platforms to concentrate receptors and assembling the signal transduction machinery; but their ability to influence the actin cytoskeleton, cell polarity, angiogenesis, membrane fusion is probably just as significant [1–20]. Fig. 1. shows a schematic view of lipid rafts, and caveolae like compositions. The lower half of the Fig. 1 depicts a typical composition of a cell death associated raft clustering enriched with ceramide (will be discussed below). All the components (lipids and proteins) presented in Fig. 1 are not available in the same type of raft. The raft composition largely depends on what fraction of lipids in the cytoplasmic leaflet form rafts in living cells and the type of cellular response after receiving signal/stimuli [2–31]. All tumor cells shed plasma membranes enriched in sphingomyelin (SM), cholesterol and gangliosides to counter possibly against hosts immune responses and keep themselves free from destruction by immune system (reviewed in ref. [1], see also [28–32]). The lipid raft proteomics is the study of all the proteins that use, and most importantly need raft assemblage for their proper functioning, certainly expressed by a given cell, tissue or organism at a given time and under specific conditions. Some of those proteins are well illustrated in the case of signaling in hematopoietic cells, including T-cells and B-cells, in a variety of cancer cells and to some extent in model rafts [1–17,19–26,33–53]. The binding of actin is an important example of interaction of raft components with cytoplasmic proteins, which implies raft mediated signaling, cell surface organization and a role for rafts in mechanical properties of cell membranes. Actin forms proteinchains such as Cadherin–Catenin–Actin, CD44–(Ezrin, Radixin, Moesin; ERM)–Actin, and some others depending on tissue and cell types where catenin and ERM-like proteins constitute a

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Fig. 1. Schematic depiction of a lipid raft. Note that, all the components shown here are not present in the same type of raft. For various types of rafts with distinct composition see references [1–6, 8–10, 13–17, 25–27]. Lower half shows typical composition of a cell death associated raft clustering enriched with ceramide when cells and tumors are exposed to radiation or challenged with therapeutic compounds. The coupling between outer and cytoplasmic leaflets is hypothetical. See also references [24, 153, 154, 224–226].

molecular bridge. Table 1 summarizes the lipid components, and Table 2 shows a few protein components of rafts as identified by biophysical, biochemical, and immuno-localization methods. All raft components cited in Table 2 are authentic and supported from

the works of at least four separate laboratories, and further confirmed by unbiased proteomics of lipid rafts [1,4,34–36,40– 45]. The proteomics approach yielded, quantified and validated around 250 proteins as authentic raft proteins [4,35–37],

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Table 1 Lipid components of rafts — their function and regulation mechanism Component lipids

Major function/(abundance in cancer with comparison to normal tissues: very high ↑↑, high↑, low ↓, or cancer stage specific↑↓)

Mechanism of control/regulation

References

Cholesterol (Chol)

Spacer between the hydrocarbon chains, H-bonding with surface water and sphingomyelin, implicated for signal transduction, precursor for steroid biosynthesis/(Yes ↑↑) Maintainance of bilayer structure, macrodomain formation, signaling/(Yes ↑) Various signaling and Immuno-protection/(Yes ↑) Signal transduction/(Yes ↑) Signal transduction/(Yes ↑) Signal transduction (↑↓)/FasL mediated apoptosis (Yes ↑↑)

Biosynthesis, efflux from and influx into cells by transport of lipoproteins cholesterol, endocytic recycling.

[1,5,8,11–17,46,52,71,119, 235–238] and references therein

Biosynthesis, and endocytic recycling

[1,5,8,11–17,22–24,46,31,119, 224–227,244] [1,5,8,11–17,28–31,49]

Sphingomyelin (SM) Glycosphingolipids (e.g., GM1) Lysophosphatidic acid (LPA) PIP2 Ceramide (Cer)

excluding the contaminations of non-raft detergent-resistant/ insoluble membrane proteins [17,38,39]. From such a large set of true raft proteins, I have picked some structural as well as signaling molecules, including cav-1, CD44, EGFR, Ras, uPAR, MMPs, and FAS, Ezrin and a few related proteins for discussion in the following sections. Manipulation of available data on lipid rafts by proteomics approach for various cancer cells and of model rafts have compelled to suggest that there may be two subsets of raft assemblage in cell membrane. One subset of raft is enriched with

Table 2 Examples of protein components of lipid rafts Marked fluctuations in the abundance of raft proteins in cancers

Swiss Prot/OMIM

References

Caveolin-1 β-Actin uPAR,

Q03135/601047 P02570 Q03405/173391

uPA

191840

β-Catenin α-Catenin CD44 FAS/CD95

116806 P26232 107269 134637

Ezrin

P26038/123900

Ras (R-Ras2, TC21) Ras-related (Rap2) Ras GAP-like IQGAP Integrin, beta 1 MMP-9 MMP-2 Receptor tyrosine kinases, EGFR Serine/Threonine kinase 35

P17082 P17964 P46940 P05556 120361 120360 O15146/131550

[1,5,8,36,46,47,51,70] [1,5,8] Reviewed in ref. [1,129–131,36,179] Reviewed in ref. [129], and [176–178] [1,88–95] [36,88–95] [1,49,76,77] [24,153,154,157,159–170, 199,224–228] [1,4,36,143,210–212, 153,154,225] [1,8,15,122] [5,8,36] [36] [1,36,130,131,133] [1,130,133,134,249] [1,130,137–141,248] [8,107,111–119]

IPI00104087.3

[5,8,36]

The raft protein itself, or one of its partners (which essentially have molecular interaction) in the signaling cascades are presented.

Biosynthesis, and endocytic recycling Biosynthesis Biosynthesis Biosynthesis, Sphingomyelin break down by ASMase

[1,8,224,227,229] [8,224,229] [19,20,22–24,119,224–227,229]

cholesterol–sphingomyeline–ganglioside (hereafter, “chol-raft”) containing proteins mainly Caveolins, CD44, and members of the RTKs family. “Chol-rafts” are responsible for cellular homeostasis, but when normal cellular signaling is dysregulated “cholrafts” promote cell transformation, tumor progression, angiogenesis and metastasis. Another subset of raft is enriched with ceramide–sphingomyeline (hereafter, “cer-raft”) containing mainly, FAS, FASL, and the members of death-inducing signaling complex (DISC). “Cer-rafts” promote apoptosis. Ezrin like molecules function as molecular bridge in both types, the “cholrafts” and the “cer-rafts”. Small rafts can sometimes be stabilized to form larger platforms through clustering of proteins, and lipid– protein–lipid raft (LPLR) reordering in living cells by elevated cholesterol induced coalescence of rafts. This reordering of “cholraft” might serve to sequester proteins namely, CD44, EGFR, Ras and stimulate “start” signals to oncogenic pathways. When cells and tumors are exposed to radiation or challenged with therapeutic compounds acid sphingomyelinase (ASMase) becomes activated. The activated ASMase then translocates to membrane surfaces (Fig. 1) and hydrolyzes SM, which generates sphingosine and ceramide. This in situ breakdown of SM elevates ceramide level, which rapidly displaces cholesterol from membrane/ lipid-“chol-raft” and forms “cer-raft”. This newly formed “cerraft” serves to sequester proteins of the FAS–DISC and related proteins, which immediately triggers “start” signals to death/ apoptosis following endocytosis. The displaced cholesterol may move to the other parts of the membrane enriched with phospholipid, and be continuously balanced by efflux of cellular cholesterol (See discussion and perspectives). 2. Lipid rafts/caveolae signaling and cancer Epigenetic regulation of genes encoding raft components and its roles in cell transformation angiogenesis, immune escape, and metastasis, and roles for rafts in other diseased states have been reviewed earlier [1,5,8,13–15,24]. The role of cholesterol and rafts in non-genomic hormonal signaling in prostate cancer is intriguing and discussed recently [25,52]. This contribution is

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devoted to focus on the cellular biochemistry and signaling function of some protein components of membrane/lipid rafts in cell transformation, tumor development and metastasis, which would help to best understanding our current concerns in raftassisted signaling in regulation of cancer, and will help to select strategies for better management of neoplasm. 2.1. Raft and caveolin-1 signaling Caveolin-1 (cav-1) protein has been identified as a marker necessary for lipid rafts/caveolae formation, stability and function, which is associated with multiple cellular processes in normal and pathophysiological state, including cancer progression and hormone refractory diseases [1,3,5,8,13–17,27,53–55]. In animal model studies targeted disruption of cav-1, and homologous recombination created cav-1-null mice have shown that natural presence of caveolae are absent from various cell types resulting in severe physical limitations in cav-1-disrupted mice. Some of those include impaired nitric oxide and calcium signaling in cardiovascular system, causing aberrations in endothelium-dependent relaxation, contractility, and maintenance of myogenic tone. In addition, cav-1 knockout mice lung displayed thickening of alveolar septa caused by uncontrolled endothelial cell proliferation and fibrosis [56,57]. Endothelial cav-1 and caveolae are necessary for both rapid and long-term mechanotransduction in intact blood vessels [53,58,61–63]. Lack of caveolae formation is associated with degradation and redistribution of cav-2, defects in the endocytosis of albumin (a caveolar ligand and a major plasma protein that transports and scavenges metabolites and endotoxic substances respectively), and a hyperproliferative phenotype in tissues and cultured embryonic fibroblasts from the cav-1-null mice [57,59]. Schubert et al. [60] has recently examined the role of "non-muscle" caveolins (cav-1 and cav-2) in skeletal muscle biology and found that skeletal muscle fibers from male cav-1(−/−) and cav-2(−/−) mice show striking abnormalities, such as tubular aggregates, mitochondrial proliferation/aggregation, and increased numbers of M-cadherin-positive satellite cells, which became more pronounced with ageing. Interestingly, they found that cav-2deficient mice displayed normal expression levels of cav-1, whereas cav-1-null mice exhibited an almost complete deficiency in cav-2. Hence, these skeletal muscle abnormalities seem to be due to loss of cav-2. Histologic examination and echocardiography identified a spectrum of characteristics of dilated cardiomyopathy in the left ventricular chamber of the cav-1deficient hearts, including an enlarged ventricular chamber diameter, thin posterior wall, and decreased contractility [61]. Cav-1 knockout (KO) mice were observed to be completely devoid of caveolae. Lewis lung carcinoma cells implanted into cav-1 KO mice had increased tumor microvascular permeability, angiogenesis, and growth [62]. Cav-1 plays a crucial role in the mechanisms that coordinate lipid metabolism with the proliferative response occurring in the liver after cellular injury came from an outstanding report that treatment of cav1-null mice with glucose, which is a predominant energy substrate when compared to lipids, drastically increased survival and reestablished progression of the cell cycle [63].

2.2. Tumor suppression and metastasis promotion. The cav-1, CD44, E-cadherin paradoxes Cav-1 functions as a membrane adaptor to link the integrin α-subunit to the Fyn tyrosine kinase. Upon integrin ligation, Fyn is activated and binds to SHC, via the SH3 domain of Fyn. SHC is subsequently phosphorylated at Y317 and recruits GRB2 (Table 3). This sequence of events is necessary to couple integrins to the Ras–ERK (extracellular signal-regulated kinase) pathway and promote cell cycle progression [64]. Mutations in cav-3 suggested that heritable differences in the interaction between caveolins and their partners may lead to other conditions, which mainly cause limb–girdle muscular dystrophy (Reviewed in ref. [8]). Cell culture and biochemical findings evidenced that cav-1 is a tumor suppressor gene and a negative regulator of the v-Src, H-ras, Protein kinase A, PKC-isoforms, and Ras-p42/44 mitogen activated protein (MAP) kinase cascade within caveolae ([8,46] and references therein). Loss of heterozygosity analysis implicates chromosome 7q31.1, (where cav-1 gene is localized to a suspected tumor suppressor locus D7S522), in the pathogenesis of multiple types of human cancer, including breast, ovarian, prostate, and colorectal carcinoma, as well as uterine sarcomas and leiomyomas (reviewed in refs. [8,65], see also [66]). For example, cav-1 expression in mammary adenocarcinoma (MTLn3) cells inhibits epidermal growth factor (EGF)stimulated lamellipod extension and cell migration, blocks their anchorage-independent growth, and thus induces a non-motile phenotype by blocking the EGF-induced activation of the p42/ 44 MAP kinase cascade [66]. Table 3 Cav-1, CD44, E-cad and FAS interacting proteins in lipid rafts / caveolae mediated signaling Proteins

References, Link: http://www.ncbi.nlm.nih.gov/ entrez/dispomin.cgi?id

Caveolin-1 Integrin α-subunit H-ras/Raf-1 Fyn tyrosine kinase Ras-p42/44 MAP kinase cascade SHC EGF/EGFR GRB2 Cyclin D-1 Src-like kinases V-Src PKA/PKC/PKC-α

[64,104,108]; 600560 [5,52,66,101,102,104]; 131530/131550 [64]; 108355 [67]; 168461 [1,5,8,49,65], 124095 [8,46]; 190090 [48]; 601639, 609191/176982/176960

CD44 Ezrin HA (hyaluronic acid) Annexin-II Rac-1 / Rac-2

[1,4,76,77], 123900 [76,77]; see 107269 and 600826 [76,77]; 151740 [77]; 602048/602049

FAS FASL Ezrin Caspase-8 and 10

[24,153,154,199–201,227,220]; 123900 [71,72,153,200] see 123900 [153,154,199,200,226,227] 151740

[64]; 603963 [2,40,42,46]; 601619/164760 [64]; 137025 [46,66]; see the caveolin link, 601047

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On the other hand, cav-1 can also function as tumor metastasis promoting molecule which is not likely to its cell growth inhibitory function ([46] and references therein, [67]). For example, higher expression of cav-1 induces filopodia formation in lung adenocarcinoma with enhanced metastasis [46]. Fig. 2 shows that concerted function of lipid rafts and cav-1 is necessary for filopodia formation and an increase of metastatic potential of lung adenocarcinoma cells. Cav-1 induces filopodia formation with enhanced metastatic potential when lipid component of rafts virtually remains unaltered. Delipidation/cholesterol depletion disrupts cav-1 function and stopped filopodia formation even when cav-1 protein was expressed in very high amount in cav-1+ve CL1-5 cells (Fig. 2B), C6 cells (Fig. 2C). Cav-1 expression is absent, or very weak in the low invasive lung cancer cell line CL1-0, which is in best agreement with the observations that down regulated cav-1 expression facilitates cell transformation [1,46,47,62,66,67]. Down-regulation of cav-1 is also associated with its 5′-proximal promoter CpGisland hypermethylation and tumor formation (reviewed in ref. [1]). Elevated cav-1 level is associated with lung, breast, prostate, and their lymph node metastases (see the link: http://www. ncbi.nlm.nih.gov/entrez/dispomin.cgi?id=176807), strengthening the possibility that cav-1 may also act as an oncogene [1,46,68]. Thompson and colleagues [68–70] have demonstrated that cav-1 expression is significantly increased in primary and metastatic human prostate cancer after androgen ablation therapy, cav-1 is secreted by androgen-insensitive prostate cancer cells, and that this secretion is regulated by steroid hormones and the overall results established cav-1 as an

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autocrine/paracrine factor that is associated with androgen-insensitive prostate cancer. It has also been suggested that cav-1 might be a therapeutic target in the case of prostate cancer. Thus evidences are accumulating in favour of cav-1 as a direct mediator of steroid action, signaling through PI3K–protein kinase B (PKB, also designated as Akt) pathway, and metastasis of cancers [1,25,71]. Akt/PKB is a serine/threonine kinase that is a critical regulator for cell survival and proliferation, especially in human malignant cancers. Activated Akt phosphorylates pro-apoptotic proteins, thereby inactivating their activities. Akt activation also up-regulates anti-apoptotic genes such as Bcl-XL and FLICE-inhibitory protein (FLIP). Akt activation involves phosphorylation of S473 and T308 by phosphoinositide-dependent kinases and integrin-linked kinase. Recent studies have suggested that rafts have implications in Akt activation [71]. Puzzling question arises, why a tumor suppressor gene whose inactivation is necessary for cell transformation and tumor induction can be re-expressed to facilitate tumor progression? Such a paradox is not restricted only to cav-1. It is apparent that other molecules, including E-cadherin, CD44, granulocyte/ macrophage-colony stimulating factor (GM-CSF), RAR-β2, and α- and β-catenins have also been reported to impart the virtual opposite function in tumorigenesis ([46], reviewed in refs. [1] and [72]). The promoters of the various cell surface adhesion marker genes, including cav-1, E-cad and CD44 are inactivated in association with promoter CpG-hypermethylation at the onset of tumor development and remains thereafter methylated in full-blown tumors but were found to be re-expressed in metastatic foci and lymph nodes. Since these genes are mostly

Fig. 2. Concerted function of lipid rafts and Caveolin-1 is necessary for filopodia formation and increase of metastatic potential of Lung Adenocarcinoma cells. High expression of cav-1 induces filopodia formation with enhanced metastatic potential when lipid component of raft virtually remains unaltered. Delipidation/cholesterol depletion disrupts cav-1 function and stopped filopodia formation even when cav-1 protein was expressed in very high amount. Cav-1+ve CL1-5 cells (B), C6 cells (C), and C7-Dox(−) cells (E) showed abundant filopodia formation. The cav-1−ve CL1-0 (the parental cells for C6 cells, A) and C7-Dox(+) cells (D) did not reveal filopodia formation in cultures. However, the ability to form filopodia in CL1-5, C6 and C7-Dox(−) cells was abolished when cells were cultured in delipidated medium (F,G, and H respectively). A rhodamine–phalloidin staining was used to highlight the presence of F-actin in filopodia and at the peripheral cytoplasmic area. Adapted with permission from Ho et al. [46].

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inactivated by DNA methylation; their reactivations certainly need demethylation activity [1,47,72–74]. For instance, mRNA and protein expression of cav-1 is frequently lost in multiple cancers. At the cancer onset, cav-1 gene is repressed by DNA methylation, while re-expression occurs just before metastasis [1,72–74]. CD44 is known to be involved in re-organization of highly dynamic structures of cytoskeleton when cells respond to extracellular stimuli by division and/or changes in shape or activity [1,13,75–77]. CD44 is the major cell surface receptor for hyaluronic acid (HA), co-localizes with annexin-II in the cell surface lipid raft microdomains in their cytoplasmic face in fibroblasts and blood cells. Both CD44 and annexin-II can be released from these lipid rafts by sequestration of cholesterol with altered cytoskeleton, prevention of Rac1, and prevention of Rac1-induced lamllipodia outgrowth, which was activated by HA binding to CD44 [76,77]. mRNA and protein expression of CD44 is frequently lost in multiple cancers at the early stage of cell transformation and tumor progression, which also is associated with DNA hypermethylation [1,72,78–81]. Again, reexpression of CD44 gene was found necessary for metastatic diffusion of many tumors [1,72,81–85]. The transmembrane glycoprotein E-cad, a calcium-dependent cell–cell adhesion molecule, is known to play a key role in the maintenance of tissue integrity by forming complexes with catenins (α, β, and γ). E-cad is eventually tagged to actin cytoskeleton through catenins. Because loss of E-cad expression results in disruption of cellular clusters, it has been postulated that E-cad functions as tumor suppressor gene. mRNA and protein expression of E-cad is frequently lost on DNA methylation in multiple cancers at the early stage of tumor progression [1,72,81,86–94]. Also in this case, reexpression of E-cad had been shown to be clinically significant at the metastatic foci of many cancers [1,72,74,85,91,92,95]. Cavalli et al. [95] have studied the epigenetic features in breast sentinel lymph nodes (SLN) compared with their corresponding match primary tumors for CpG-island hypermethylation of a few genes, including THBS2, E-Cad, and RAR-β2 in six paired primary breast tumors and their matched SLN. They noticed that, overall, 71% (30/42) of the methylation measurements were identical between the primary tumors and the SLN. Dissection of such alterations may lead to identification of initial events associated with the metastatic dissemination process. 3. Raft and epidermal growth factor receptor (EGFR) signaling Cohen (1962) first described a growth factor that has a profound effect on the differentiation of specific cells in vivo, and which is a potent mitogenic factor for variety of cultured cells of both ectodermal and mesodermal origin that is known today as epidermal growth factor (EGF) or Urogastrone (URG). URG/EGF (OMIM 131530) is also a potent inhibitor of gastric acid secretion and promotes epithelial cell proliferation. Mature EGF is a single-chain polypeptide consisting of 53 amino acids and having a molecular mass of about 6 kDa only [96,97]. The EGF receptor (EGFR) is a 170 kDa transmembrane lipid raft glycoprotein comprising a 1186 amino acid polypeptide chain and is composed of three domains: an extracellular ligand-

binding domain, a single transmembrane lipophilic region, and an intracellular domain that exhibits intrinsic tyrosine kinase activity [98–104]. The extracellular ligand-binding region on the cell membrane is connected to the intracellular machinery that possess the tyrosine kinase activity via a single hydrophobic membrane domain [98,99]. Helin et al. have demonstrated that the biological activity of the human EGFR is positively regulated by its C-terminal tyrosines [100]. Endogenous ligands to EGFR include TGF-α, heparin-binding EGF, amphiregulin and betacellulin [105]. It transmits signals upon activation by complex formation with the cognate ligand, EGF, or in some instances activated by cross-talk with the other ligands of receptor tyrosine kinases (RTKs) family, namely TGF-α [97–99]. On binding of the ligand to EGFR, the ligand– receptor complex undergoes dimerization and internalization [104,106,107]. The EGFR family plays an essential role in normal organ development by mediating morphogenesis and differentiation, and plays a crucial role in growth, differentiation, and motility of normal as well as cancer cells. Yamabhai and Anderson [102] have identified that the second cysteine-rich region of EGFR contains targeting information for caveolae/rafts. Puri et al. [104] have shown that several endocytic proteins are efficiently recruited to morphologically distinct plasma membrane lipid rafts, upon activation of EGFR. Analysis of detergent-resistant membrane fractions revealed that the EGF-dependent association of endocytic EGFR proteins with rafts is as efficient as that of signaling effector molecules, such as GRB2 or SHC [104,108]. Specialized membrane microdomains have the ability to assemble both the molecular machineries necessary for intracellular propagation of EGFR effector signals and for receptor internalization. In the case EGFR the localization of the two processes is well characterized ([104] and references therein). Signaling occurs within specialized membrane microdomains, lipid rafts [5,13], whereas endocytosis occurs mostly through the clathrin-coated pits [104,106,109]. The signal transduction pathways can lead to cell proliferation and tumor growth, as well as progression of invasion and metastasis [98,105,107,110]. Fig. 3 (right half) shows a schematic view for how EGFR signaling cascade operates and where “chol-rafts/caveolae” are involved. Overexpression of EGFR produces a neoplastic phenotype in tumor cells, which is associated with higher rates of progression from superficial to invasive forms of various cancers [1,66,105,107,111–114]. Nagane et al. [115] have reported that a common mutant EGFR confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis. Thomas et al. [116] have reported that cross-talk between G protein-coupled receptor (GPCR) and EGFR signaling pathways contributes to growth and invasion of head and neck squamous cell carcinoma (HNSCC). Combined blockade of both EGFR and GPCRs may be a rational strategy to treat cancers, including HNSCC that shows cross-talk between GPCR and EGFR signaling pathways [116]. Scartozzi et al. [117] have analyzed the expression of activated (phosphorylated) Akt and MAPK in 98 cases of paired primary colorectal tumours and metastases with the aim to define better the EGFRrelated molecular profile of colorectal cancer (CCR) as a tool for

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treatment selection. EGFR downstream signaling pathway can be hyper activated even in the absence of EGFR expression in a considerable proportion of patients, which indicates cross-talk signaling. Curto et al [118] have shown that upon cell–cell contact neurofibromatosis type 2 (NF2) tumor suppressors, Merlin, coordinates the processes of adherens junction stabilization and negative regulation of EGFR signaling by restraining the EGFR into a membrane compartment from which it can neither signal nor be internalized. The (n-3) fatty acids (FA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) decrease proliferation and induce apoptosis in MDA-MB-231 human breast cancer cells. Schley et al. [119] have examined the effects of EPA and DHA on the lipid composition of lipid rafts as well as raft localization of EGFR, and phosphorylation of EGFR. EPA and DHA treatment decreased lipid raft sphingomyelin, cholesterol, and diacylglycerol content; and in the absence of linoleic acid, both EPA and DHA increased ceramide levels in raft. Furthermore, there was a marked decrease in EGFR levels in

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lipid rafts, accompanied by increases in the phosphorylation of both EGFR and p38 MAPK, in EPA + DHA-treated cells. As sustained activation of the EGFR and p38 MAPK had been associated with apoptosis in human breast cancer cells, their results indicate that (n-3)-FA modify the lipid composition of membrane rafts and alter EGFR signaling in a way that decreases the growth of breast tumors. The result is intriguing and shows antithetic effects of EGFR signaling on cell regulation. Oh et al. [120] have investigated whether membrane cholesterol could regulate apoptosis, and elucidated a mechanism by which apoptosis is induced in prostate cancer cells. When LNCaP cells were exposed to 2-hydroxypropyl-beta-cyclodextrin (HPCD), cell viability was inhibited by HPCD dose dependently, and restored by replenishment of cholesterol. Caspase-3 and PARP cleavage assays suggested that cells were dying on application of HPCD by apoptotic cell death through down-regulation of Bcl-XL, and inhibition of both EGFR/Akt and EGFR/ERK signal transduction pathways, which indicated the lowering of the pool of “chol-raft”.

Fig. 3. Raft signaling to survival and death. Right panel: Lipid rafts/Caveolae can break the MAP kinase pathway at multiple points. Growth factor receptors, e. g., EGFR (a “chol-raft” protein) initially trigger recruitment of the GRB2/Sos complex to the plasma membrane leading to sequential activation of Ras, Raf, MEK and Erk, which translocates to the nucleus to phosphorylate Elk-1 and other transcription factors. See the text for details. Left panel: FAS-ligation is comparatively a slow process, and rate of which reaction depends on the types of signals cells receive. However, once FAS–FASL complex is formed, FAS interacts with Ezrin very fast for internalization, which may be the rate limiting step. At the same time transformation of “chol-raft” to “cer-raft” is over, for which involvement of ASMase is necessary. Internalization of FAS–DISC in association with “cer-raft” leads to death. See the text for details and compare with Fig. 4.

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4. Nuclear factor kappa B signaling The oxidative stress sensitive transcription factor playing critical roles in the regulation of a variety of genes, important in multiple cellular responses, is the nuclear factor-kB (NFκB). NFκB remains inactive in the cytoplasm sequestered through its interaction with IkB and became activated in inflammation and cancer. IkB kinase phosphorylates IkB and subsequent ubiquitination cause degradation of the latter. Eventual release of NFκB is followed by its translocation to the nucleus. The NFκB inducing kinase controls activation of IkB kinase, followed by a cross-talk between activation of the extracellular signal-regulated kinase (ERK)/MAP kinase pathway, and the NFκB-inducing kinase/IkB kinase/NFκB pathway. NFκB is reported to be directly associated with lipid rafts by Misra et al. [121]. They have observed that, in T-cells, following TCR ligation, a small portion of total cellular caspase-8 and c-FLIPL rapidly migrate to lipid rafts where they associate in an active caspase complex. Activation of caspase-8 in lipid rafts is followed by rapid cleavage of c-FLIPL at a known caspase-8 cleavage site. The active caspase/ c-FLIP complex forms in the absence of FAS and recruits the NFκB signaling molecules RIP1, TRAF2, and TRAF6, as well as upstream NFκB regulators PKC, CARMA1, Bcl-10, and MALT1, which connect to the TCR. Inhibition of caspase activity, furthermore, blocks NFκB activation. These findings define a link among TCR, caspases, and the NFκB pathway that occurs in a sequestered lipid raft environment in T-cells [121]. Disruption of lipid rafts might deregulate the above kinase-axis, since MAP kinase function depends largely on its association with lipid rafts/caveolae [8,46,57,58] (See Fig. 3). Because based on recent studies, NFκB is considered as a target for the management of cancer, modulation of this pathway by targeting lipid rafts could also contribute to its preventive potential (See Discussion and perspectives for further information). 5. Rafts in MAP kinase, Ras, and activator protein 1 (AP1) signaling MAP kinases have been shown to play important roles in many cellular physiologic processes, including proliferation, differentiation, and survival or death. In mammalian cells there are the three major types of MAP kinases: (i) c-Jun NH2terminal kinases (JNK), (ii) p38 MAP kinases, and (iii) ERK. Activation of ERK1 and ERK2 (ERK 1/2) in this pathway modulate a wide variety of cellular activities via the regulation of several transcription factors. The ability of the ERK/MAPK pathway to promote cell growth by activation of cyclin D is counterbalanced by the concomitant production of the cyclindependent kinase inhibitor p21WAF1. Moderate activation of the pathway leads to cell proliferation, while hyperactivation results in p21WAF1-mediated growth arrest. In addition, induction of the cell cycle inhibitory INK4 proteins, including p16INK4A , is mediated by the Ras/Raf/MEK/ERK pathway (Fig. 3, right half). Several studies have implicated that overexpression and activation of ERK/MAPK plays an important role in colon cancer progression, which may be a useful molecular target for colon cancer therapy.

The major pathways that lie down stream of the membraneassociated receptor tyrosine kinases (RTK) is activation of Raf-1 by lipid raft associated Ras [5,122], which follows phosphorylation mediated activation of MAPK/ERK kinase 1/2 (MEK1/2). Activated MEK1/2 then phosphorylates ERK1/2. The JNK1/2/3 and p38α/β/γ pathways are parallel MAP kinase cascades in mammalian cells [46,121–125]. Once activated, MAP kinases (ERK, JNK and p38) activate nuclear transcription factor E-26-like protein (ELK) and c-Jun [123,126]. Phosphoinositide 3-kinase (PI3K) is activated by RTKs and it synthesizes the second messenger, phosphatidyl inositol-3,4,5-triphosphate, which is necessary for phosphorylation of Akt (protein kinase B). Akt directly phophorylates and inactivates the pro-apoptotic protein Bad, and enhances the antiapoptotic function of Bcl-XL. In LNCaP human prostate cancer cell line, it was shown that lipid rafts mediate Akt-regulated survival [1,123,127]. Raf-1 also is a component of lipid rafts, and because the deregulated overexpression of MAP kinase pathway is frequently seen in a variety of human cancers, modulation of MAP kinases by disruption of lipid rafts along with the use of some natural compound, therapeutic agents and other inhibitors, including MEK1/2 inhibitor PD98059 (2-(2′-amino-3′-methoxyphenyl) oxanaphthalen-4-one), and the PKC-δ inhibitor rottlerin may provide novel strategies for the prevention and treatment of cancer. AP-1 transcription factor is a heterodimer of leucine zipper super family proteins, specially, heterodimer of the c-Jun and c-Fos proteins. High AP-1 activity has also been shown to be involved in the tumor induction and progression of various types of cancers, including colon, lung, breast, skin and prostate cancers ([123] and references therein). 6. Raft and insulin like growth factor mediated signaling Insulin like growth factor (IGF) family of ligands, associated proteins and its receptors is a significant essential growth factor system involved in the maintenance of cellular functions. Mastick et al [128] have shown that insulin stimulates the tyrosine phosphorylation of caveolin. The coupling of free IGFs to IGF-1 results in intracellular receptor autophosphorylation and phosphorylation of precise downstream targets, which leads to cross activation of several signaling pathways, including PI3K/Akt pathway and the Ras/MAP kinase pathway, both of which have components docked in lipid rafts (Table 2). This leads to activation of cell proliferation by inducing specific genes and DNA replication. Therefore targeting the IGF-1 signaling pathway through lipid rafts might be an effective strategy for prevention and cure of few cancers (reviewed in refs. [5,123], see also [128]). 7. Extracellular matrix and raft signaling The ability of cells to respond appropriately to environmental cues is critical to maintaining cellular, tissue and organism homeostasis. One such environmental cue is derived from cellular adhesion to the extracellular matrix (ECM). The loss of adhesion-dependent cellular regulation can lead to increased cellular proliferation, decreased cell death, changes in cellular differentiation status, and altered cellular migratory

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capacity; all of which are critical components of cellular carcinogenesis and metastasis of the disease [28–32,125,129–135]. 8. Rafts in MMPs and uPAR signaling The invasive nature of tumor cells vexed and bedeviled the current treatment pathways, as the remaining tumor cells inevitably invade the surrounding normal tissues, which leads to tumor recurrence. Such local invasion remains to be an important cause of mortality and underscores the need to understand in depth the mechanisms of invasion. Several proteases influence the malignant characteristics of various carcinomas, gliomas and sarcomas — their inhibition could prove to be a useful therapeutic strategy [1,129–135]. Extracellular matrix is a key component of the tissue destroyed by tumor-cell invasion. This is a dynamic environment that has a pivotal role in regulation of cellular functions during normal and pathologic remodeling processes, such as embryonic development, tissue repair, inflammation, and tumor invasion and metastasis. Protease profiling studies have indicated that expression of the serine protease urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) an authentic lipid–raft protein [1,36], and expression of the cysteine protease cathepsin-B, and of the matrix metalloproteinases MMP2 and MMP9 is increased in high-grade cancers, including prostate, breast, and astrocytomas compared with respective low-grade prostate, breast, and astrocytomas, and the normal prostate, breast, and brain [129–134]. Strategies to prevent the expression of uPA and uPAR at the molecular level have led to significant reduction/inhibition of tumor invasion and growth. Down-regulation of MMP2 and MMP9 expression through approaches such as MMP inhibitors or antisense vectors results in less tumor cell invasion and the inhibition of tumor growth and angiogenesis. Aberrant, persistent inclusion into lipid rafts limits the tumorigenic function of MMP1 in malignant cells (reviewed recently, [1]). Studies with cathepsin-B using its natural inhibitor, cystatin-C and antisense vectors have shown significantly to reduce tumor formation and invasiveness. These proteases may have interacted with each other and directly or indirectly can facilitate the expression of other proteases, as such; the inactivation of one molecule seems to cause reduced expression of the other molecule and/or the entire pathways [51–54,121–123,129–136]. The progression of human tumors involves the MMP family. Two particular members of the family, MMP2 and MMP9, seem to have an important role in invasion and metastasis of varied tumors [30,129–134,137– 141]. DNA methylation has also been shown to affect MMPs expression [140]. uPA is a trypsin-like protease that converts the zymogen plasminogen into active plasmin. It has the ability to prevent apoptosis, stimulate angiogenesis, mitogenesis, cell migration, and to modulate cell adhesion. Inhibition of urokinase can decrease tumor size or even complete remission of cancer in mice. The known urokinase inhibitors are highly toxic and need high concentration for effective inhibition. It has been reported by molecular modeling that tea polyphenol EGCG blocks urokinase activity by interacting with H57 and S195 extending toward R35

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from a positively charged loop of urokinase thus prevents the formation of the catalytic triad of urokinase [123,129,142]. Thus destabilization of uPAR function by disturbing its raft assembly may help blocking of uPA activity, one of the most frequently overexpressed enzymes in human cancers. 9. Rafts in integrin and focal adhesion kinase mediated signaling The focal adhesion kinase (FAK) family kinases (which include FAK and pyk2) regulate cell adhesion, migration, and proliferation in a variety of cell types ([136], reviewed in refs. [143,144]). Adhesion of cells to the ECM is mediated by heterodimeric transmembrane integrin receptors located within sites of close apposition to the underlying matrix called focal adhesions. Integrin engagement and clustering stimulates FAK phosphorylation on Y397 , creating a high affinity binding site for Src and Src family kinases, the premier lipid raft associated family of kinases [1,5,13,135,136,143–146]. The FAK/Src complex phosphorylates many components of the focal adhesion, resulting in changes of adhesion dynamics and the initiation of signal cascades. In addition to FAK catalytic activity, FAK also functions as a scaffold to organize structural and signaling proteins within focal adhesions. The importance of FAK as a regulator of normal cellular function is underscored by the number of cancers reported to have alterations in FAK expression and/or activity, including prostate, colon, breast, cervical, ovarian, head and neck, colon, liver, stomach, sarcoma, glioblastoma and melanoma, and in addition alterations in FAK expression and/or activity have been associated with tumorigenesis and increased metastatic potential [136,143–146]. Currently, it is unclear how the catalytic activity and/or scaffolding function of FAK contribute to tumor progression. To date studies of FAK function relied on expression of dominant interfering mutants or elimination of FAK expression by gene knockout, repression by antisense oligonucleotide or siRNA. A recent report on the biochemical and cellular characterization of a novel small molecule inhibitor, PF573,228 that targets FAK catalytic activity shows that the inhibitor interacts with FAK (recombinant or endogenous) in the ATP-binding pocket and bloks its phosphorylation at Y397 position in a variety of normal and cancer cell lines [136]. Babuke and Tikkanen [147] recently have reviewed the role of reggie and flotillin (flot) in various cellular processes such as insulin signaling, T-cell activation, membrane trafficking, phagocytosis, and EGFR signaling. Reggie-1/flot-2 and reggie-2/ flot-1 are ubiquitously expressed and their molecular functions depend on post-translational modifications such as phosphorylation and lipid modifications. Hazarika et al. [148] have demonstrate that overexpression of flot-2, alters the phenotype of SB2 melanoma cells and is associated with up-regulation of the GPCR for thrombin, PAR-1. Although flot-2 is also expressed in normal melanocytes, these data suggest that the levels of flot-2 increase with melanoma progression in cell lines and in melanocytic lesions. PAR-1 is upstream to the B-Raf/MAPK/ ERK pathway and implicated in melanoma progression. One might expect that flot-2 may influence other GPCR, so that

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modulation of PAR-1 by flot-2 may have significance for signal transduction beyond this specific tumor model [147–149]. 10. Raft signaling and apoptosis Programmed cell death (apoptosis) is a highly ordered protective mechanism through which unwanted, fatigued or damaged cells are eliminated from the system, which is essential for normal development, immunological competence, and homeostasis. Moreover, apoptotic cell death, which is preceded by the activation of effector proteases, known as caspases, which results in the cleavage of varied endogenous proteins involved in structure maintenance and signal transduction in an organism. Also, apoptosis serves as a protective mechanism against neoplastic developments by eliminating genetically damaged cells or excess cells that have improperly been induced to divide. It is characterized by marked changes in cellular morphology, including chromatin condensation, membrane blebbing, nuclear breakdown and the appearance of membrane associated apoptotic bodies, internucleosomal DNA fragmentation, cleavage of poly(ADP-ribose) polymerase (PARP), as well as by release of hypoacetylated and trymethylated histone H4 [122,132,150–156]. For many cancers, including lung, cholangiocarcinoma, and cancer of endothelial cells when in advanced malignant stage, treatment/management options become restricted to radiation therapy and/or chemotherapy. Current data on the molecular mechanisms of radiation/chemo-induced tumor cell killing suggest that tumor cell destruction follow apoptotic pathway through transformation of “chol-raft” to “cer-raft”, and “cer-raft”mediated FAS–DISC internalization. 11. FAS and raft mediated FAS signaling cDNA encoding the Human FAS antigen (TNFRSF6/FAS/ APT1/APO1/CD95, OMIM 134637) consist of a 16-amino acid signal sequence followed by a mature protein of 319 amino acids with a single transmembrane domain and a molecular mass of approximately 36 kD [157]. The protein contains three domains; a FAS death domain, a FAS ligand (FASL) binding domain, and the transmembrane domain. The FAS antigen shows structural homology with a number of cell surface receptors, including tumor necrosis factor (TNF) receptors and the low-affinity nerve growth factor receptor (NGFR). Northern blot analysis detected 2.7 kb FAS mRNAs in thymus, liver, ovary, and heart. Functional expression studies in mouse cells showed that the FAS antigen induce antibody (FASL)-triggered apoptosis. Canale and Smith [158], reported defect in the FAS gene in a patient with autoimmune lymphoproliferative (lpr) syndrome (ALPS; OMIM, 601859), which was due to mutation. Drappa et al. [159] confirmed that in the patients affected son, who had a heterozygous 972G-to-T transversion within the death domain of the FAS gene, resulting in a D244Y substitution. Subsequently, it had been shown that the FAS receptor can rapidly be expressed on T-cells following activation of T-cell hybridomas, and that the interaction between FAS and FASL-induced cell death occurs in a cell-autonomous manner consistent with

apoptotic features [160,161]. Mannick et al. [162] have demonstrated that FAS activates caspase-3 by inducing the cleavage of the caspase zymogen to its active subunits and by stimulating the denitrosylation of its active site thiol. MYCinduced apoptosis requires interaction between FAS and FASL on the cell surface [163]. The findings of Hueber et al. linked the two apoptotic pathways, establishing the dependence of MYC on FAS signaling for its killing activity. The signaling pathway leading to apoptosis by FAS cross-linking with FASL results in the formation of a death-inducing signaling complex (DISC) composed of FAS, the signal adaptor protein FAS-Associated via Death Domain (FADD), and procaspase-8 and 10, and the caspase-8/10 regulator c-FLIP [164,165]. This association generates Casp8/10, activating a cascade of caspases. LeppleWienhues et al. [166] have demonstrated that in addition to the role of FAS in inducing cell death, stimulation of FAS inhibits the influx of calcium normally induced by activation of the T-cell antigen receptor, in part, by not affecting the release of calcium from intracellular stores. This block in calcium entry can be mimicked by stimulating T-cells with acid sphingomyelinase (ASMase) metabolites of the plasma membrane lipid sphingomyelin, such as ceramide and sphingosine [166]. Grassme et al. [167] have showed that Pseudomonas aeruginosa infection induced apoptosis of lung epithelial cells were by activation of the endogenous FAS/FASL system. Deficiency of FAS or FASL on epithelial cells prevented apoptosis of lung epithelial cells in vivo as well as in vitro. Many studies have indicated that perhaps all tumor cells express FAS, but with mutations. However, Arscott et al. [168] have demonstrated that the FAS antigen is expressed and functional in papillary thyroid cancer cells. Lee et al. [169] have analyzed the entire FAS coding region by microdissection techniques of biopsy samples from 21 burn scar-related squamous cell carcinomas (BSRSCC) with somatic point mutations in all of the splice sites from 3 patients. The FAS mutations were located within the death domain, ligand-binding domain, and transmembrane domain. No mutations were detected in 50 cases of conventional SCC [169]. Loss of heterozygosity (LOH) of the other FAS allele was demonstrated in tumors carrying the N239D and C162R mutations, and expression of FAS was confirmed in all tumors with FAS mutations. BSRSCC is usually more aggressive than conventional SCC, and Lee et al. have suggested that somatic mutations in FAS may contribute to the development and/or progression of BSRSCC and identified following in the FAS gene: a 957A-to-G transition resulting in an N239D substitution in the FAS death domain; a 547A-to-G transition resulting in an N102S substitution in the FAS ligand-binding domain; a 726T-to-C transition resulting in a C162R substitution in the FAS transmembrane domain. Zhang et al. in 2005 have genotyped 1,000 Han Chinese lung cancer (211980) patients and 1,270 controls for 2 functional polymorphisms in the promoter regions of the FAS and FASL genes, -1377G-to-A (134637.0021) and -844T-to-C (134638.0002), respectively. Compared to non carriers, there was an increased risk of developing lung cancer for carriers of either the FAS -1377AA or the FASL -844CC genotype; carriers of both homozygous genotypes had a more than 4-fold increased risk [170]. Their results support the hypothesis

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[171], that the inactivation of FAS- and FASL-triggered apoptosis pathway plays an important role in human carcinogenesis. In animal model studies of lpr mice showed that FAS is the gene for the mouse lpr, and is identical to human patients displaying a phenotype similar to that of lpr mice [172,173]. The murine phenotype autosomal recessive lpr was characterized by lymphadenopathy, hypergammaglobulinemia, multiple autoantibodies, and the accumulation of large numbers of nonmalignant CD4-, and CD8-T-cells. Affected mice usually developed a systemic lupus erythematosus-like autoimmune disease, and a defect in the negative selection of self-reactive T lymphocytes in the thymus. Autoimmune disease in mice may be due to integration of endogenous retrovirus in the FAS gene [174]. The importance of FAS in the pathogenesis of diabetes was evaluated by generating non-obese diabetic mice that develop spontaneous autoimmune diabetes, with beta cell-specific expression of a dominant-negative point mutation in the FAS death domain [175]. In vivo silencing effect of siRNA duplexes targeting the FAS gene to protect mice from liver failure and fibrosis in two mouse models of autoimmune hepatitis, has indicated that FAS siRNA specifically reduced FAS mRNA and protein levels in mouse hepatocytes which were resistant to apoptosis [176]. It is observed that FAS-deficient lpr −/− mice had less severe collagen-induced arthritis, possibly by blocking the IL1R1 or TLR4 (603030) pathway that is generally activated by FAS–FASL interaction [177]. Landau et al. have pointed that FAS-deficient lpr mice developed a Parkinson's disease phenotype [178]. Fisher et al. [179] have identified a heterozygous mutation in the FAS gene in 5 unrelated children (134637.0001–134637.0005), with a rare ALPS. The disorder was characterized by massive nonmalignant lymphadenopathy, autoimmune phenomena, and expanded populations of TCR–CD3(+)CD4(−)CD8(−) lymphocytes, and each child had defective FAS-mediated T lymphocyte apoptosis in vitro. One of the patients studied by Fisher et al. was included in the report by Sneller et al., who were delineating this disorder and pointed out its resemblance to autosomal recessive lpr/ gld (generalized lymphoproliferative disorder) disease in the mouse. The lpr and gld mice bear mutated genes for FAS and FASL, respectively [180]. Many other investigators have identified frame shift mutations due to base deletions or insertions in various exons coding regions, LOH as well as single nucleotide polymorphism of the FAS gene from ALPS (type IA, IB, II and III) patients having multiple phenotypic features of ALPS, including elevated numbers of double-negative T-cells, hypergammaglobulinemia, and FAS mutations were found in a fraction of CD4+ and CD8+ T-cells, monocytes, and CD34+ hematopoietic precursors, and B-cell lymphoma ([169,181–188], for an exciting review see [189]). Siegel et al. have found dominant interference of FAS mutations in the first cysteine-rich domain that results ligandindependent molecular interaction of wild type and mutant FAS receptors through a specific region of the extracellular domain, rather than depending upon FASL-induced FAS oligomerization, in a preassociated receptor complex which normally permits FAS signaling [188–190]. Epigenetic DNA-methylation studies on FAS expression and sensitivity to its ligation on murine CD8 cells specific for the

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CW3 antigen expressed by transfected P815 cells implicated that loss of FAS expression by antigen-specific cytotoxic T-cells may be due to DNA methylation [191]. In an attempt to evaluate the potential role of FAS gene as a model of gene therapy Shimizu et al. [192] have evaluated genetic and epigenetic events leading to alternation of the introduced FAS gene. They noticed solid tumors formed by FAS cDNA-transfected hepatoma cells, F6b, were almost completely cured by a single treatment of anti-FAS monoclonal antibody (mAb) but recurred in gld/gld–lpr/lpr mice after initial complete response. Recurred tumors were resistant to repeated mAb treatment. The FASresistant tumor contained two types of cells without FAS protein, and with decreased FAS protein. Cells having no FAS were due to the deletion of FAS cDNA. However, FASdecreased cells retained FAS cDNA, which was highly methylated. Petak et al. [193] have demonstrated that DNA hypermethylation is one mechanism that contributes to the downregulation of FAS expression and subsequent loss of sensitivity to FAS-induced apoptosis in colon carcinoma cells. Impaired function or repression of FASL may inactivate FAS stimulation and facilitate tumor growth. Castellano et al. [194] have shown that active transcription of the human FASL/TNFSF6 promoter region in T lymphocytes was involved in chromatin remodeling in association with DNA methylation. The resistance of small cell lung cancer (SCLC) cells by FASL and TRAIL induced apoptosis has been explained by an absence of FAS and TRAIL-R1 mRNA expression and a deficiency of surface TRAIL-R2 protein [195]. In addition, caspase-8 expression was absent, whereas FADD, FLIP and caspases-3, -7, -9 and -10 were detectable. Loss of mRNA and protein expression was well correlated with DNA methylation of the respective genes encoding FAS, Casp-8 and TRAIL-R. Peli et al. [196] have nicely demonstrated that the oncogenic potential of H-ras may reside on its capacity not only to participate in the mitogen activated signaling cascade to promote cellular proliferation (Fig. 3, right panel), but also to simultaneously inhibit FAS-triggered apoptosis. They reported that oncogenic Ras (H-Ras) downregulated FAS mRNA and protein expression in association with DNA methylation, and rendered cells of fibroblastic and epitheloid origin resistant to FASLinduced apoptosis. They suggested that Ras signals via the PI3kinase pathway to downregulate FAS, suggesting that the known anti-apoptotic effect of the downstream PKB/Akt kinase may be mediated, at least in part, by the repression of FAS expression. Many neuroblastomas have hypermethylation and down-regulation of CASP8, leading to resistance to TRAIL. van Noesel et al. [197] have analyzed methylation of multiple genes in 22 neuroblastoma cell lines. In 40% neuroblastoma cells FLIP gene adjacent to CASP8 map at chromosome 2q33 was methylated. Down-regulation of FLIP strongly corresponds with down-regulation of Casp-8, and this was also found for DCR1 and DCR2. The FLIP protein is a negative regulator of Casp-8, and its methylation patterns showed a moderate correlation. Co-methylation patterns were observed for the TRAIL receptor pairs DCR1 and DCR2, and between DR4 and DR5. All four receptors co-localize in chromosome band 8p21. The hypermethylation status of the FAS promoter in prostatic and bladder carcinomas and respective cell lines appears to play a role

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in down-regulation of FAS expression, which might be one of the causes of tumor formation in prostate and bladder [198]. The ability of FAS signaling to mediate death signals and to play a critical role in development pathways may hinge on its ability on downstream caspase activation. Two reasonable candidates for mediating the regulation of signaling consequences may be c-FLIP, and ASMase. C-FLIP at high concentrations can inhibit FAS-mediated apoptosis ([199] and references therein). Another possible switch between pro-apoptotic and non-apoptotic FAS signaling may be the post-translational modifications that regulate the ability of FAS to become internalized after its activation by FASL. Two acceptor sites for post-translational modifications in the intracellular domain of FAS may have importance in apoptotic activities of FAS. C199 regulates two prerequisite steps for FAS internalization, localization of the receptor to lipid rafts and FAS aggregation [200], and Y291 is important for internalization of FAS [201]. Simultaneously, ASMase translocation to lipid raft may cause ceramide accumulation by catalytic hydrolysis of SM to ceramide and sphingosine. This locally produced ceramide remodels raft to form larger domains of “cer-raft” and promotes rapid aggregation of FAS as well as FAS–FASL complex formation [20,22–24,166]. Internalization of FAS into lipid raft or an endosomal compartment may determine which signaling pathways are involved. When internalization of FAS is blocked, the receptor cannot induce apoptosis and instead remains fully engaged, may be, in activating non-apoptotic pathways [201]. FAS, therefore, seems to be similar to other internalizing receptors such as the EGFR. Internalization of EGF target receptors, EGFR, and EGFR downstream receptor sequestration in the lipid rafts or endocytic compartment may thus contribute to both the intensity of signaling and assembly of signaling complexes [104,202]. 12. Rafts and ezrin signaling Ezrin (OMIM 123900) is a component of the microvilli of intestinal epithelial cells that serves as a major cytoplasmic substrate for certain protein–tyrosine kinases. It is the same as cytovillin (CVL), which is a microvillar cytoplasmic peripheral membrane protein that is expressed strongly in placental syncytiotrophoblasts and in certain human tumors. cDNA cloning, sequencing, and deduction of protein sequence indicated that human ezrin is a highly charged protein with an overall pI of 6.1 and a calculated molecular mass of 69 kDa, which is close to serum albumin [59,203,204]. A 3.2-kb ezrin mRNA is known to express the protein with a relative highest level in intestine, kidney, and lung cells. The cDNA clone hybridized to DNAs from widely divergent organisms, indicating that the sequence is highly conserved throughout evolution. Within its N-terminal domain, ezrin showed a high degree of similarity of amino acid sequence to the erythrocyte cytoskeletal protein band 4.1 (OMIM 130500). Saotome et al. [205] have found that conditional mutation in the ezrin gene gave birth of homozygous mutant mice at sub-Mendelian ratios (about 12%), but despite their normal appearance at birth they failed to thrive and did not survive past weaning. The luminal surface of ezrin −/− intestine was covered with cauliflower-shaped villi that appeared to be

aggregates of multiple individual villi. They observed that the morphologic complexity of the ezrin −/− villi increased with postnatal age, leading to the formation of complex amalgamated structures, but the establishment and maintenance of epithelial polarity was not affected. Hence, it was concluded that ezrin is not absolutely required for the formation of brush border microvilli, but it performs a critical function in organizing the apical domain of the intestinal epithelial cell and its associated apical junctions [205]. Ezrin, radixin (RDX; OMIM 179410), and moesin (MSN; OMIM 309845), the so-called ERM proteins, act as linkers between the plasma membrane and the actin cytoskeleton. They are involved in a variety of cellular functions, such as cell adhesion, migration, and the organization of cell surface structures. They are highly homologous, both in protein structure and in functional activity, with merlin/schwannomin, the NF2 tumor suppressor protein [206]. Ezrin is predominantly a raft protein [4,36]. Gupta et al. [4] have recently analyzed B-cell lipid rafts by quantitative proteomic analysis, and B-cell raft proteome reveals that ezrin regulates antigen receptor-mediated lipid raft dynamics. Roumier et al. [207] has demonstrated that ezrin, F-actin (see ACTA1; OMIM 102610), and CD43 (OMIM 182160) relocalize to the sides, not the center, of the T-cell-(Antigen Presenting Cell) contact area after formation of immunologic synapse, which suggested that ezrin may contribute to setting the scaffold between the actin cytoskeleton and transmembrane proteins facilitating cell–cell interactions and receptor retention. Using cells from Cd43 −/− mice, Allenspach et al. [208] observed that ERM proteins move independently of the large CD43 mucin. Overexpression of a dominant-negative ERM mutant containing the N-terminal 320 amino acids of ezrin inhibited the activation-induced movement of CD43 without affecting conjugate formation. The dominantnegative mutant reduced cytokine production but not the expression of T-cell activation markers. By double staining of ezrin and palladin (OMIM 608092) in several cell lines, Mykkanen et al. [209] have found that the subcellular localization of ezrin differed between epithelial and smooth muscle cells. In epithelial cells, such as HeLa, ezrin localized at the cortical actin skeleton and demonstrated little overlap with palladin. However, in intestinal smooth muscle cells, ezrin demonstrated a filamentous staining pattern and partial colocalization with palladin. Yu et al. [210] have established highly and poorly metastatic rhabdomyosarcoma cell lines derived from a transgenic mouse model overexpressing Hgf/Sf (142409) and deficient for Ink4a/Arf (OMIM 600160), in which skeletal muscle tumors reminiscent of those in embryonic rhabdomyosarcoma (OMIM 268210) arise with very high penetrance and short latency [211]. Yu et al. (2004), then used cDNA microarray analysis of these cell lines to identify a set of genes whose expression was significantly different between highly and poorly metastatic cells. Subsequent in vivo functional studies revealed that ezrin and Six1 (OMIM 601205) have essential roles in determining the metastatic fate of rhabdomyosarcoma cells. VIL2 and SIX1 expression was enhanced in human rhabdomyosarcoma tissue, significantly correlating with clinical stage [211]. By imaging osteosarcoma cells in the lungs of mice, Khanna et al. [212] showed that ezrin expression provides an early survival advantage for cancer cells

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that reach the lung. AKT and MAPK3 (OMIM 601795) phosphorylation and activity were reduced when ezrin was suppressed. Ezrin-mediated early metastatic survival was partially dependent on the activation of MAPK but not AKT and availability of CD44. To define the relevance of ezrin in the biology of metastasis beyond the founding mouse model, Khanna et al. have examined ezrin expression in dogs that naturally developed osteosarcoma. High ezrin expression in dog tumors was associated with early development of metastases. Consistent with this data, they found a significant association between high ezrin expression and poor outcome in pediatric osteosarcoma patients [212]. 13. Acid sphingomyelinase and raft signaling Acid sphingomyelinase (OMIM 607608) is a lysosomal sphingomyelin phosphodiesterase (EC 3.1.4.12). Stress is believed to activate sphingomyelinase to generate ceramide, which serves as a second messenger in initiating apoptotic response. The first conclusive evidence for this paradigm was provided by Santana et al. [213], who showed that lymphoblast from Niemann–Pick patients failed to respond to ionizing radiation with ceramide generation and apoptosis. Earlier, Suchi et al. [214] have demonstrated that the metabolic defect in cultured Niemann–Pick disease cells, which lack sphingomyelinase activity, could be corrected by retroviral-mediated transfer of human ASMase cDNA. Targeted disruption of the Smpd1 gene (human homologue of SMPD1 gene) in transgenic mice was achieved by homologous recombination in embryonic stem cells [215]. Homozygous mice accumulated sphingomyelin extensively in the reticuloendothelial system of liver, spleen, bone marrow, and lung, as well as in the brain. Most strikingly, the ganglionic cell layer of Purkinje cells of the cerebellum degenerated completely, leading to severe impairment of neuromotor coordination. The picture resembled that of the neurovisceral form of Niemann–Pick disease type A. Horinouchi et al. [216] obtained similar results in Smpd1 knockout mice. Using different human epithelial cells and primary fibroblasts, Grassme et al. [217] demonstrated an activation of phospholipase C and ASMase in TNF-α (FAS)-mediated hepatocellular apoptosis induced by Neisseria gonorrhoeae, resulting in the release of diacylglycerol and ceramide. Genetic and/or pharmacologic blockade of ASMase and phosphatidylcholine-specific phospholipase C caused inhibition of cellular invasion by the pathogen. GarciaBarros et al. [23] have studied that endothelial apoptosis is a homeostatic factor regulating angiogenesis-dependent tumor growth. Moreover, microvascular damage regulates tumor cell response to radiation at the clinically relevant dose range. In recent years ASMase has emerged as a biochemical mediator of stimuli as diverse as ionizing radiation, chemotherapeutic drugs, UVA light, heat, FAS activation, reperfusion injury, as well as infection with some pathogenic bacteria and viruses [22–25,218–228]. ASMase activity is also crucial for developmental programmed cell death of oocytes [23,24]. A comprehensive model has been proposed for a role of ASMase to explain the function ceramide plays in FAS-induced apoptosis. Upon contacting the relevant stimuli, ASMase translocates into and generates ceramide within distinct plasma membrane “chol-rafts”. The in situ produced ceramide displaces

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cholesterol and transforms “chol-raft” to “cer-raft” (Compare Figs. 1, 3, and 4). Fig. 4 shows a scheme representing new molecular steps occurring upstream of FAS internalization involving rafts. Posttranslational palmitoylation of FAS (A) allows the targeting of the FAS receptor to lipid raft (precisely, “chol-raft”) (B), where following stimulation (for examples, radiation or chemotherapeutic drug) FASL binds to FAS and ASMase translocates to the raft and generates ceramide by hydrolysis of SM and transforms “chol-raft” to “cer-raft”, “cerrafts” coalesces to larger rafts which eventually sequesters FAS–FASL complex and the connection between FAS receptor and actin cytoskeleton occurs via the association of FAS with ezrin (C). The ezrin mediated cytoskeleton association initiates “cer-raft”–FAS–FASL internalization (D), a prerequisite step for the intracellular optimal formation of DISC (E), which leads to an efficient caspase activation and cell death (see also refs. [24,199,200]). DeMorrow et al. [222] have recently investigated the mechanism of actions of two endocannabinoids, namely, anandamide and 2-arachidonylglycerol, on regulation of cholangiocarcinoma growth. It was observed that although anandamide was antiproliferative and pro-apoptotic, 2-arachidonylglycerol stimulated cholangiocarcinoma cell growth. Interestingly, target of action of both the drugs was lipid rafts. 2-arachidonylglycerol treatment effectively dissipated the lipid raft structures and caused the lipid raft-associated proteins lyn and flot-1 [1,8,147–149] to disperse into the surrounding membrane. Anandamide treatment induced an accumulation of ceramide, which was required for anandamide-induced suppression of cell growth by FAS-mediated apoptosis. Natural inhibitors of angiogenesis like, thrombospondin-I and pigment epithelium-derived factor induce FAS/FASL-mediated apoptosis and block angiogenesis without harming the preexisting vasculature. Volpert et al. have demonstrated that both inhbitors upregulated FASL on endothelial cells, thereby specifically sensitizing the stimulated cells to apoptosis by inhibitor-induced FASL. It may be considered as an example of cooperation between pro- and antiangiogenic factors in the inhibition of angiogenesis, and provided one explanation for the ability of inhibitors to select remodeling capillaries for destruction [190]. The studies documenting selective effects of lipid rafts for chemotherapeutic induction of apoptosis and cell cycle arrest for cancer cells were described by Gajate and Mollinedo [153,154,225–227], while investigating the mechanism of action of the novel anti-tumor drugs edelfosine and aplidin. They and others have discovered a potent and novel cell-killing mechanism that involves the formation of FAS-driven scaffolds in membrane raft clusters, housing death receptors and apoptosis-related molecules after application of chemotherapeutic drugs. FAS, tumor necrosis factor-receptor 1 (TNF-R1) and TNF-related apoptosis-inducing ligand (TRAIL)-R2/DR5 were clustered into lipid rafts of various cell lines, including leukemic Jurkat, M624 melanoma, and breast cancer cells following radiation or drug treatment, and the presence of FAS had been being essential for apoptosis [22–24,153,154,153,154,219–227]. Actin-linking proteins ERM-trio, RhoA and RhoGDI were also clustered into FASenriched rafts in drug-treated leukemic cells [153,154,225–228]. The functions of SM in FAS-mediated apoptosis by FAS clustering

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Fig. 4. A scheme of molecular steps involving raft that occur upstream of FAS internalization Posttranslational palmitoylation of FAS (A) allows the targeting of the FAS receptor to lipid raft (precisely, “chol-raft”) (B) where following stimulation FASL binds to FAS and ASMase translocates to the raft and generates ceramide by hydrolysis of SM and transforms “chol-raft” to “cer-raft”, “cer-rafts” coalesce to larger rafts which eventually sequester FAS–FASL complex and the connection between FAS receptor and actin cytoskeleton occurs via the association of FAS with ezrin (C). The ezrin mediated cytoskeleton association initiates “cer-raft”-receptor internalization (D), a prerequisite step for the intracellular optimal formation of DISC (E), which leads to an efficient caspase activation and cell death. Modified after adaptation with permission from Chakrabandhu et al. [200].

through aggregation in lipid rafts have nicely been demonstrated by cloning a gene responsible for SM synthesis (SMS1), and establishing SM-synthase-defective WR19L cells transfected with the human Fas gene (WR/Fas–SM(−)), and cells that have been functionally restored by transfection with SMS1 (WR/Fas–SMS1) [224]. It has been observed that production of membrane SM enhances FAS-mediated apoptosis through efficient translocation of FAS into lipid rafts, FAS clustering increasing DISC formation, activation of caspases with concomitant increase in ceramide generation within lipid rafts upon FAS stimulation [221,224]. FASmediated apoptosis involves FAS activation both in a FASLindependent way and following FAS–FASL interaction in an autocrine way, through the accumulation of FAS, membranebound FASL and signaling molecules in membrane rafts and high amount of ceramide [23,24,224–228]. 14. Discussion and perspectives Structural and functional role of lipid rafts at the plasma membrane as well as in cell organelles, including Endoplasmic reticulum and Golgi apparatus has been analyzed and reviewed in detail in several studies [1,3,5,7–17,19–24]. In recent years a specific activity of membrane sub-domains, including SM, sphingosine and ceramide has been observed to contribute to cell death by apoptosis [19–24,218–222]. Although detected in various cell types, the role of such sub-domains in apoptosis has however been mostly studied in lymphocytes and endothelial cells where the physiological apoptotic program occurs after FAS triggering and in situ generation of sphingosine and ceramide by ASMase activity [19–24,218–223]. Current data suggest that ceramide–sphingolipid rich sub-domains mimic cholesterol–

sphingolipid rich lipid rafts, and manipulation of ceramide metabolism and/or the function of ceramide-enriched membrane platforms may present novel therapeutic opportunities for the treatment of cancer, degenerative disorders, pathogenic infections or cardiovascular diseases [24,153,154,221–228]. Sphingolipid- and cholesterol enriched membrane microdomains are considered to be floating in an “ocean” of phospholipid, and hence have been termed rafts [16]. Although there is dispute in the field as to the size of these domains, it is usually safe to say that they range in size from 10 to 200 nm in diameter [9,10,18]. Recent contribution to this field is the recognition that consumption of sphingolipids within rafts to generate ceramide results in dramatic alteration of these small rafts. Ceramide results in the coalescence of sub-domains into large-membrane macrodomain. These structures serve as platforms for protein concentration and oligomerization for transmitting signals across the plasma membrane (see Figs. 1, and 3). Ceramide, which has the unique property of fusing membranes and the capability to self-associating through hydrogen bonding (reviewed by van Blitterswijk et al., [229]), appears to drive the coalescence of raft microdomains to form large, ceramide-enriched membrane platform, which exclude cholesterol [19,20,24,219–221]. Studies with model membrane, unilamellar vesicles composed of phosphatidylcholine/sphingomyelin that was locally treated with sphingomyelinase, showed that ceramide generation is followed by the formation of patches by ceramide that coalesced rapidly into ceramide-enriched macrodomain [19,20,230]. The formation of distinct domains in model membranes upon the addition of ceramide was also demonstrated by Huang et al. [231], who employed a magnetic resonance spectroscopy technique to demonstrate a transition of fluid phospholipid bilayers (non-raft)

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into a gel phase (raft-like) upon addition of ceramide. Veiga et al. [232] similarly demonstrated that ceramide partitions into distinct domains instead of mixing with phospholipids. Displacement of cholesterol by ceramide from lipid rafts decreases the association of the raft-binding and cholesterol binding proteins, for example, cav-1 [233]. Outstanding questions arise: where does the displaced cholesterol go? The displaced cholesterol may move to the other parts of the phospholipid ocean. Cellular cholesterol is continuously balanced by influx of exogenous cholesterol and efflux of cellular cholesterol. Cholesterol can be taken up from the lipoproteins of circulation by mechanism involving of desorption – transfer of cholesterol from the lipoproteins to the exoplasmic leaflet of the plasma membrane bilayer – or by receptor-mediated uptake (for detailed discussions, see Simons and Ikonen, [234]). Cellular cholesterol is continuously lost by release of cholesterol to circulating lipoproteins. Such a loss can be quite rapid, up to 0.1% of total cholesterol per minute. The release from the plasma membrane can take place by desorption of cell surface cholesterol into lipoproteins or be induced after high-density lipoprotein binding to membrane receptors. In many cells, including cancer cells mechanism of cholesterol removal is by membrane shedding, a process releasing plasma membrane vesicles that may be enriched in raft lipids [1,28–32]. Reasonably, cholesterol behaves differently with respect to efflux whether it is in rafts or non-rafts membrane matrix. Raft cholesterol is more slowly extracted by cyclodextrin and by HDL; hence, non-raft cholesterol is the most likely source for desorptive efflux. The increase in cellular cholesterol levels may lead to increased deposits of cholesterol esters (CE) in cytoplasmic lipid droplets. As a matter of fact, deposition of cholesterol was detected in various tumours almost a century ago [235–237], which also is well over a period of 60 years before the proposition of lipid bilayer membrane structure (reviewed in refs. [1,234,238]). In erythrocyte membranes, cholesterol has been found to exhibit transbilayer translocation with a half time of about 50 min, so that efficient efflux may indeed require a specific flippase. ATPbinding cassette transporter ABCA1 has also been found to promote Ca2+-induced translocation of phosphatidyl serine to the exoplasmic leaflet [239], which may favour the release of phospholipids and cholesterol to HDL. ABCA1 protein localized to both the plasma membrane and to the Golgi complex [239] and could also be involved in the transport of cholesterol form the Golgi to the cell surface, possibly involving rafts [240]. Other interesting questions: are there quantitative differences in the amount of lipid rafts, or at least lipid contents of rafts, in between normal cells and cancer cells? The number of rafts as such has not been counted and compared so far between normal and cancer cells. But, elevated levels of cholesterol-rich lipid rafts in cancer cells can be correlated with apoptosis sensitivity induced by cholesterol depleting agents and shedding of membrane vesicles from plasma membrane (PM), and data on comparisons of the lipid contents between normal cells and cancer cells are widely available. Cholesterol deposition in various tumours has been observed since many decades ago [235–237]. For instance, the clear cell form of RCC is known to derive its Histologic appearance from accumulations of glycogen and lipid. It has been

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found that the most consistently stored lipid form is CE [241]. Gebhard et al. [241] investigated that clear cell cancer (CCC) tissue contains 8-fold higher total cholesterol and 35-fold more esterified cholesterol than found in normal kidney. CE appears to be formed intracellularly since it was not membrane-bound and since oleate was the predominant form, as opposed to linoleate in lipoprotein CEs. Further, Gebhard et al. analyzed that the cholesterol in clear cell tumours may not be appeared as a result of excessive synthesis from acetate since HMG–CoA reductase activity was lower in those cancer tissues than in normal kidney (2.9+ 0.8 vs. 7.2 + 1.2 pmol/mg of protein per min). In contrast, intracellular activity of fatty acyl‑coenzyme A: cholesterol acyl transferase (ACAT) was higher in tumour tissue than in normal kidney (2405 + 546 vs. 1326+ 301 pmol/mg of protein per 20 min) while cytosolic CE activity appeared normal. CE storage in CCR and other cancers may be a result of primary abnormality in ACAT activity or it would be a reduced release of free cholesterol (relative to cell content) with a secondary elevation in ACAT activity [235–238,241]. It has been proposed that progressive increases in membrane cholesterol contribute to the expansion of rafts/caveolae, which may potentiate oncogenic pathways of cell signaling [1,25,27,46,52,71,127,238]. In a growing tumour the cells need new blood vessels to take up nutrients and clean up offal. Neo-formed blood vessels may also become pathways for metastasis, circulation of cancer cells at distant organs and tissues from the full-blown tumour (reviewed in ref. [1]). Activated cells shed fragments of their plasma membrane into the extracellular milieu, and the processes of shedding are particularly evident in actively growing tumour cells that continually need shedding of membrane vesicles, in vitro and in vivo. Extracellular membrane vesicle(s) from tumour cells (EMVTCs) derived from selected areas of plasma membrane analyzed and appear to be enriched with rafts components: cholesterol, GM1, GM3 and SM, ceramide, and contain surface antigens and proteases often present in tumour cells. [31,242– 246]. Dahiya et al. [244] have observed that there are differences in lipid composition in human prostatic adenocarcinoma cell lines, and that the SM level was significantly higher in highly metastatic cells (DU-145 and ND-1) compared with the lower metastatic variant (LNCaP). van Blitterswijk et al. [245] have found that the lipid fluidity in purified PM of murine leukemic GRSL cells, as measured by fluorescence polarization, is much higher than in PM of normal thymocytes. In contrast, significant differences were found between PM and EMVTCs from GRSL cells. In this system a much lower lipid fluidity of the shed EMVTCs was found, due to the much increased cholesterol/ phospholipid molar ratio (3.5-fold) and SM (9-fold) content, as compared to the PM. Several proteases, including MMPs are enriched in EMVTCs/ rafts and thought to play a role in tumour cell invasion and metastasis [31,32,245–247]. Endothelial cell migration and invasion through ECM are essential for neo-vascularisation, and EMVTCs/rafts enriched with SM and MMP2/MMP9 was found capable to increase the process by 3–5 folds [31,248,249]. Addition of purified EMVTCs and SM independently in culture media may promote the formation of capillary-like structures of cancer cells [31]. Tumour cells produce various cytokines and chemokine that

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attract various leukocytes – neutrophils, eosinophils, macrophages, dendritic cells, and mast cells, as well as lymphocytes – all of which are capable of producing a spectrum of cytokines and cytotoxic mediators including reactive oxygen species, serine and cysteine proteases, MMPs, membrane perforating agents, and soluble mediators of apoptosis, such as TNF-α, interleukins and interferons, IFNs [1,32,130–132,247–249]. In addition to altering the local balance of pro-angiogenic factors during melanoma development and in at least human cervical, breast and prostate carcinogenesis, angiogenesis was found to be activated in mid stage lesions, prior to the appearance of full-blown tumours (reviewed in refs. [1,131]). The progression of human tumors involves the MMP family. Two particular members of the family, MMP2 and MMP9, seem to have an important role in invasion and metastasis of varied tumors [30,129–134,137–141]. Chetty et al. [250] have discovered that down-regulation of MMP2 by adenovirus-mediated delivery of MMP2 siRNA (Ad-MMP2) reduced spheroid invasion and angiogenesis in vitro, and metastasis and tumor growth in vivo. Ad-MMP2 infection led to the induction of apoptosis in A549 lung cancer cell lines. Ad-MMP2 decreased the content of the antiapoptotic members of the Bcl-2 family proteins and increased the content of the pro-apoptotic members of the Bcl-2 family. Furthermore, Ad-MMP2-mediated apoptosis was accompanied by increase in truncated Bid, release of cytochrome-C and the activation of caspase-8, -9 and -3. Ad-MMP2 infection caused up-regulation of FAS–FASL and FADD, and Anti-FASL antibody reversed Ad-MMP2-induced apoptosis. Tissue inhibitor of metalloproteinases (TIMP)-3, an endogenous inhibitor of MMP2, which cleaves FASL and activates the FAS–FASL -inducing apoptotic pathway, was increased in Ad-MMP2-treated cells. Adenovirusmediated expression of MMP2 siRNA in human lung xenografts in vivo resulted in increased FAS, FASL, cleaved Bid and TIMP3. This show that MMP2 inhibition upregulates TIMP3 levels, which in turn, promotes apoptosis in lung cancer. Barnhart et al. [251] have demonstrated that despite their apoptotic potential, human tumors may be refractory to the cytotoxic effects of FASL. Hence, tumour progression may be promoted by non-apoptotic signals emanating from FAS that include (but are not limited to) activation of NFκB and all three major MAPK pathways: ERK1/2, p38 and JNK1/2. Misra et al. [121] have defined a link among TCR, caspases, and the NFκB pathway that occurs in a sequestered lipid raft environment in Tcells. Watabe et al. [252] have demonstrated that caffeic acid phenethyl ester (CAPE), known to inhibit NFκB, induce apoptosis via FAS-signal activation in human breast cancer MCF-7 cells. CAPE activates FAS by a FASL-independent mechanism, induces p53-regulated Bax protein, and activates caspases. The expression of dominant-negative c-Jun, which inhibits the JNK signal, also suppresses CAPE-induced apoptosis, suggesting MAPKs are involved in CAPE-induced apoptosis. The expression of FAS-antisense oligomers significantly suppressed the CAPE-induced activations of JNK and p38 and apoptosis as compared with FAS sense oligomers. These phenomena are attributable to the inhibition of NFκB by CAPE, as examined by the effect of a truncated form of IκBα (IκBδN) lacking the phosphorylation sites essential for NFκB activation. IκBδN expression not only inhibited NFκB activity but also induced

Fas activation, Bax expression, and apoptosis. These findings demonstrate that NFκB inhibition is sufficient to induce apoptosis and that FASL-independent activation of FAS plays a role in NFκB inhibition-induced apoptosis in MCF-7 cells. 14.1. Tumour suppression and metastasis promotion duality A protein that is known to have dual function may exert distinct function through lipid rafts. Biochemical analyses in cultured cells have established that cav-1 is a tumor suppressor gene and a negative regulator of the v-Src, H-ras, protein kinase A, PKC-isoforms, and Ras-p42/44 MAPK kinase cascade within caveolae ([8,46] and references therein). Upon withdrawal of cav-1 signal the rate of cell proliferation would overtake the rate of apoptosis, thereby cause higher growth of the tumor through lipid raft regulation of PI3K/Akt pathway [1,8,25,46–49,65–71]. Alternatively, cav-1 may also act as metastasis enhancing protein, which is unlikely to its growth suppression properties [46,67]. For example, filopodia formation with enhanced metastasis in lung adenocarcinoma is associated with higher expression of cav-1 [46]. As shown in Fig. 2, a concert between lipid rafts and cav-1 function is necessary for filopodia growth and increase of metastatic potential of lung adenocarcinoma cells. The picture emerges from such data is that the tumour cells use some reversible molecular programme for tumour growth and cancer metastasis. Two common strategies for shifting the balance may be involved: firstly, altered composition of membrane domains; and secondly, altered gene transcription. One of the many ways of altering membrane domains may be the transition of “chol-raft” to “cer-raft” and inactivation of proteins like, cav-1. Since cav-1 is predominantly a “chol-raft” marker, cav-1 may not signal through “cer-raft” [233]. This is, likely, being the case with UVradiation and carcinogen induced tumorigenesis, where formation of ceramide may also play the secondary role for ceramide as second messenger (for outstanding discussion of ceramide as second messenger, see [229]). But this hypothesis needs more experimental support. The second concept, altered gene transcription, may favorably be argued as many raft-component genes, including cav-1 are epigenetically regulated by dynamic flux of methyl layer at cytosine-5′C of promoter-CpG-islands of DNA. CpG-island methylation is one of many epigenetic switches, and play critical roles in controlling expressions of raft proteins involved in tumour growth and cancer metastasis [1]. Some other genes, including CD44, E-cad, EGFR, FAS, MMP 2, MMP 9, and uPAR may be regulated by many epigenetic switches, including DNA methylation and histone modifications [1,72–74,78–95,191–198,253–255]. Inhibition of histone modification enzyme, histone deacetylase, has been implicated for remodeling lipid rafts [256,257]. Lipid raft proteomics dictates various types of exchanges in between intracellular compartments and intercellular cell–cell, guest–host vicinity, including enzymes like, uPA, MMPs; cytokines and motility factors that modify the surrounding ECM [1,4,35–37,39–45,128–134,258,259]. Raft association is essential for proteins like, H-ras, one of the major components of oncogenic signals to be transported [1,5,8] to the site of action to exert its biological activity [5,67,260,261]. Raf-1,

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a component of lipid rafts, is a downstream regulator of MAPK pathway (Fig. 3). Because the deregulated overexpression of many proteins of MAPK pathway is frequently seen in a variety of human cancers, modulation of MAP kinases by disruption of “chol-raft” along with the use of some natural compound, therapeutic agents and other inhibitors, including MEK1/2 inhibitor PD98059, and the PKC-δ inhibitor, rottlerin, may provide novel strategies for the prevention and treatment of cancer. One of the oncogenic signals is transmitted by Akt also. Akt activation up-regulates anti-apoptotic genes such as Bcl-XL and FLIP [199,262–268], and recent studies have suggested that rafts are involved in Akt activation [71,262,269–272]. In correlation with Akt inactivation and Bcl-XL down-regulation, methyl-beta-cyclodextrin (MBCD) induces apoptosis through a mitochondrial pathway, including mitochondrial membrane potential change and casp-3 activation. It is intriguing that EGF administration could not restore Akt activation once rafts/ caveolae were disrupted, but cholesterol addition may reactivate Akt in the absence of EGF. Consistent with these phenomena, cholesterol repletion but not EGF administration rescued cells from the apoptosis induced by raft/caveola disruption [273,274]. Internalization-defective oncogenic EGFR are known to reside in caveolae even after ligand binding [104,108], and this prolonged EGFR activity in caveolae generates altered signaling pathways such as the enhanced tyrosine phosphorylation of the caveolae molecules, cav-1 and dynamin [64–66,275]. Assuming that the macromolecular complexes exhibit reduced diffusion in biological membranes, Lajoie et al. [276] have reported that competition between the galectin lattice and oligomerized cav-1 microdomains for EGFR recruitment regulates EGFR signaling in tumor cells. In mammary tumor cells deficient for Golgi beta1, 6N-acetylglucosaminyltransferase V (Mgat5), a reduction in EGFR binding to the galectin lattice allows an increased association with stable cav-1 cell surface microdomains that suppresses EGFR signaling. Depletion of cav-1 enhances EGFR diffusion, responsiveness to EGF, and relieves Mgat5 deficiencyimposed restrictions on tumor cell growth. In Mgat5(+/+) tumor cells, EGFR association with the galectin lattice reduces first-order EGFR diffusion rates and promotes receptor interaction with the actin cytoskeleton. Importantly, EGFR association with the lattice opposes sequestration by cav-1, overriding its negative regulation of EGFR diffusion and signaling. Therefore, they concluded that cav-1 is a conditional tumor suppressor whose loss is advantageous when beta1,6GlcNAc-branched N-glycans are below a threshold for optimal galectin lattice formation [276], which provides a rational supportive explanation for dual function of cav-1, and inactivation of cav-1 in “cer-raft” enriched membrane domains [233]. A construct of B82L cells transfected with noninternalizing oncogenic EGFR underwent apoptosis after MBCD treatment indicating that the cholesterol depletion can induce apoptosis in oncogenic pathways involving EGFR inactivation [262]. FAS-, caspase 8-, and caspase 3-dependent signaling is suggested to fold properly the raft assembly by separating non-raft lipids, including aminophospholipid and phosphatidylserine, through activation of aminophospholipid translocase and phosphatidylserine externalization in human erythrocytes [277].

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Molecular modeling with tea polyphenol EGCG and uPA has revealed that, EGCG blocks urokinase activity by interacting with H57 and S195 extending toward R35 from a positively charged loop of urokinase, which thus prevents the formation of the catalytic triad of urokinase [123,129,142]. Interestingly, Adachi et al. [278] have demonstrated in HT29 colon cancer cells that, EGCG can alter the lipid order of the membrane during EGFR inhibitory activity of EGCG. Their findings suggest that EGCG inhibits the binding of EGF to the EGFR by altering membrane raft organization, which prevents the EGFR dimmer formation. It also may explain the ability of EGCG and similar phytochemical to inhibit activation of other membrane associated RTKs, and that they may play a critical role in the anticancer effects [278]. Finally, the picture becomes clear that there are two types of functional rafts in living cells: rafts enriched with cholesterol– sphingomyeline–ganglioside, recruit proteins like caveolins, CD44, and members of the RTKs family, designated as “cholrafts” (Fig. 1, upper portion); and rafts enriched with ceramide– sphingomyeline, prefer proteins like FAS, FASL, Ezrin, and other members of FAS–DISC, designated as “cer-rafts” (Fig. 1, lower portion). “Chol-rafts” are responsible for cellular homeostasis, but when normal cellular signaling is dysregulated “chol-rafts” promote cell transformation, tumor progression, and metastasis (See Fig. 3); and “cer-rafts” promote endocytosis of the FAS–DISC leading to apoptosis, but when dysregulated “cer-rafts” may also contribute to cell transformation, tumor progression, and metastasis. Small rafts, either “chol-raft” or “cer-raft” may sometimes be stabilized to form larger platforms through clustering of proteins, and be endocytosed by LPLR reordering in living cells to extinguish the specific signals. Elevated cholesterol induces also coalescence of rafts, which serve to sequester proteins, which are activated after receiving stimuli, including CD44, EGFR, Ras and stimulate “start” signals to various cellular pathways, including tumorigenesis. When cells and tumors are exposed to radiation or challenged with therapeutic compounds ASMase becomes activated. The activated ASMase then translocates to membrane surfaces and drags more and more SM near in the vicinity (Figs. 1, and 3) and hydrolyzes SM generating sphingosine and ceramide. This in situ produced ceramide rapidly displaces cholesterol from membrane/lipid-“chol-raft” and generates “cer-raft”. These newly formed “cer-rafts” cause endocytosis of the FAS–DISC and helper proteins, which immediately extinguish the signals to death/apoptosis (See Fig. 3, and 4). Here, I have given an overview of how lipid rafts are involved in cell signaling either for proliferation or for apoptosis. Signals for controlled or enhanced proliferation are transduced via caveolae protein, cav-1 and “chol-raft”-containing proteins of the RTKs family (for example, EGFR); whereas signals for apoptosis are transduce through “cer-raft”-containing proteins of the FAS–DISC (for example, FAS itself). The single most notable feature of rafts is their participation in so many aspects of cell biology, ranging from the fundamental (for example, cell polarity) to the highly specialized (for examples, angiogenesis and immune escape). In the future there will no doubt be many more surprises and examples of cellular controls by these molecular assemblages. The analyses of individual raft proteome of

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lipid rafts have provided numerous instances where lipid rafts influence the signaling function of other cell membrane receptors. Lipid rafts can either inhibit or activate other rafts, in some cases this results in intrinsic modulation of cell fate. These effects may account for the potency of lipid rafts antagonists for inhibiting angiogenesis. Lipid rafts most often endocytosed with growth factor as well as death-receptor in signaling pathways to enhance their respective activity. Detailed analysis of the MAP kinase and FAS pathways has revealed multiple points of intersections, suggesting a complex network of interactions coordinating cell survival and death. The importance of cytoskeletal organization and mechanical tension for raft mediated signaling thereby provides a mechanism by which these considerations can influence the responses to growth factors as well, providing a convergence point for different classes of extracellular cues. Lipid rafts can also cause ligand-independent trans-activation of tyrosine kinase growth factor receptors like, EGFR, and death receptors like, FAS. Effects of this type have the potential to broaden the range of rafts signals but, when uncontrolled, result in tumorigenesis. The picture emerging from these studies is that lipid rafts function as nodes within webs of signaling, adhesive, cytoskeletal, proliferative and apoptotic pathways. These complex networks enable lipid raft-mediated participation of individual proteins in highly regulated processes such as migration, growth control and morphogenesis. But like all systems greater complexity also affords greater opportunities for subversion. Understanding these circuits in more detail should shed light on a variety of pathologic states; including cancer, impaired immune function, atherosclerosis, Parkinson's disease, as well as would shed light on intrinsic robustness of homeostasis. Acknowledgement I apologise for many other important contributions that I have not been able to include and discuss. References [1] S.K. Patra, S. Bettuzzi, Epigenetic DNA methylation regulation of genes coding for lipid raft-associated components: a role for raft proteins in cell transformation and cancer progression (Review), Oncol. Rep. 17 (2007) 1279–1290. [2] A.M. Davey, R.P. Walvick, Y. Liu, A.A. Heikal, E.D. Sheets, Membrane order and molecular dynamics associated with IgE receptor cross-linking in mast cells, Biophys. J. 92 (2007) 343–355. [3] K. Jacobson, O.G. Mouritsen, R.G. Anderson, Lipid rafts: at a crossroad between cell biology and physics, Nat. Cell Biol. 9 (2007) 7–14. [4] N. Gupta, B. Wollscheid, J.D. Watts, B. Scheer, R. Aebersold, A.L. DeFranco, Quantitative proteomic analysis of B cell lipid rafts reveals that ezrin regulates antigen receptor-mediated lipid raft dynamics, Nat. Immunol. 7 (2006) 625–633. [5] K. Simons, D. Toomre, Lipid rafts and signal transduction, Nature Reviews / Mol. Cell Biol. 1 (2000) 31–41. [6] M. Dykstra, A. Cherukuri, H.W. Sohn, S.J. Tzeng, S.K. Pierce, Location is everything: lipid rafts and immune cell signaling, Annu. Rev. Immunol. 21 (2003) 457–481. [7] S.K. Patra, A. Alonso, F.M. Goni, Detergent solubilizations of phospholipid bilayer in the gel state: the role of polar and hydrophobic forces, Biochim. Biophys. Acta. 1373 (1998) 112–118.

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