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The keystone symposium on lipid rafts and cell function, which brought together several scientists in the field of biophysics, biochemistry, and cell biology defined "membrane rafts as small (10-200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions. 20''. Eggeling et al., 2009 research work, employed Stimulated emission depletion far-field fluorescence correlation spectroscopy (STED-FCS) to detect single diffusing (lipid) molecules in nanosized areas in the plasma membrane of living cells, have also confirm that lipid nanodomains are located within <20nm diameter area21. Despite the fact that rafts are dynamic, they may arise from the stabilization of otherwise transient, nanoscale domains, often in an actin-dependent manner. Lipid rafts are separated from other membrane domains by line tension26. The author explained that Line tension refers to the energy required to create the boundary between a domain (referred to hereafter as a raft) and the surrounding membrane. Also, she stated that rafts practically noted to be thicker than the surrounding membrane, thus providing hydrophobic mismatch and henceforth, contributing to the energy required to maintain it as separate phase26. The author in addition discussed that several studies have showed that the greater the difference in thickness between the two phases, the higher the line tension and this is associated, in turn, with the formation of larger rafts. Proteins that have raft affinity, include: glycosylphosphatidylinositol (GPI)-anchored proteins, doubly acylated proteins, cholesterol-linked and palmitoylated proteins such as Hedgehog, and transmembrane proteins, particularly palmitoylated ones27. Lingwood et al., 2009 in their review paper explained that protein-based heterogeneity joins with cholesterol-sphingolipid assemblage-potential to functionalize rafts in living organisms. Thus, according to them rafts assemblages are wherein bioactivity is dependent on lipid and protein governed by standard biochemical ligand interactions, i.e. hydrogen bonds, van der Waals attractions, hydrophobic/hydrophilic interactions and electrostatic forces19. In cell membranes attractive forces between sphingolipids and cholesterol mediate the formation of lateral lipid clusters in an unsaturated glycerolphospholipid environment28.
Integrins are cell surface receptor molecules, which bind to components of the extracellular matrix (ECM). With at least 25 distinct pairings from 8Î² and 18Î± subunits29. Integrin conserved structure consist of both large extracellular domain, a short transmembrane and a short cytoplasmic domain with usually 20 to 50 amino acids, except for Î²4 which has a large cytoplasmic domain of more than 1000 amino acids30. Kuphal et al., 2005 have grouped integrins into four subfamilies including: Î²1 that function in linking cells to ECM molecules; Î²2 and Î²7, which mediates interactions to intercellular adhesion molecules (ICAMs), E-cadherin and fibrinogen; Î²3 playing a significant role in increasing tumorigenicity in malignant melanocytes, blood coagulation; and the Î±v subfamily is involve in mammalian organogenesis. Integrins have also been characterized based on their binding to specific receptors namely: RGD receptors, leukocyte-specific receptors, collagen receptors and laminin receptors31. Generally, an excess of Î² subunits exist in the cell and the amount of Î± subunit determines the amount of receptor that will go to the cell surface, henceforth free Î± and Î² subunits do not exist at the cell surface32. There are basically two members of the Î²3 integrin subfamily namely: Î±IIÎ²3 and Î±vÎ²3. Switala-Jelen et al., 2004 reviewed that Î±v tail is composed of three Î²-sandwich domains: an Ig-like "thigh" domain and two "calf" domains. They also explained that the Î²3 tail is built of a PSI (plexins, semaphorins, integrins) domain, four epidermal growth factor (EGF) domains, and a Î²-tail domain (Î²TD)33. Î±vÎ²3 is canonically known to recognize the following ligands denatured collagen, fibronectin, fibrinogen, laminin, matrix metalloproteinase-2, osteoponin, prothrombin, thrombospondin, Von Willebrand factor, vitronectin etc. They fundamentally play a role in cell-cell and cell-ECM interactions. Integrin Î²3 has been identified to be associated with certain histopathological characteristics that predict increased tumorigenicity of malignant melanocyte30. Î±IIÎ²3 plays significant role in platelet aggregation and thrombus formation, meanwhile, Î±vÎ²3 is involve in invasive malignant cells and in tumor endothelial 33.
The Structural Composition And Activation Of Src
Src is a nonreceptor tyrosine kinase of molecular weight 60 kDa. Src family kinases generally consists of eight members, categorized into two subfamilies, those that are Lyn related (Lyn, Hck, Lck and Blk) and those that are Src related (Src, Yes, Fyn and Fgr). Also, there are members outside this group that are identified as SFK related kinases including; Brk, Frk and Srm. The eight family members share similar domain arrangement with an N-terminal special region (50-70 residues) that varies among the family members, however, usually encompasses a myristoylation and sometimes palmitoylation site34. This is followed by approximately 50 amino acid Src homology 3 (SH3) that is required to direct definitive association with proline rich motifs related to the PXXP consensus34. There is also approximately 100 amino acid Src homology 2 (SH2) domains, which provides interaction with phosphotyrosine motifs. Last but not least, domain is the kinase (âˆ¼300 residues), or Src homology 1 (SH1), responsible for the enzymatic activity34. The structure of Src has been discussed at great length in Boggon and Eck, 2004 review paper. The authors explained that Src kinases basically consist of four major domains namely: the unique region as the name implies distinguish the different members of the family; followed the SH3, SH2 and SH1 domains35. The N-terminal site is myristoylated playing a vital role in signal transduction, whereas, the C-terminal site is phosphorylated at Tyr 527 enabling its binding with the SH2 domain to keep the Src kinases in an autoinhibitory state35. The SH3 domain binds the linker segment between the SH2 and kinase domain, which forms a polyproline type II helix keeping the Src in an autoinhibitory state35. SH2 domain has a conserved Arg 175 that is important for phosphotyrosine recognition. The SH1 domain also referred to as the kinase domain has an A-loop that can be phosphorylated at Tyr416 to bring about autophosphorylation in the Src kinases. The SHI domain is kept in check by the Trp 260 and Tyr 527 at its both ends to prevent the Src kinases from becoming constitutively active35. Wang et al., 2005 employed ï¬‚uorescent resonance energy transfer FRET-based Src reporter, which facilitated the visualization and quantiï¬cation of the mechanoactivated Src with high temporal and spatial resolution in live human umbilical vein endothelial cells. Their research has showed that Src can be locally mechanically activated through integrin mediated mechanism and long-range by cytoskeleton36. Src upon activation is autophosphorylated at tyrosine 419.
THE ROLES OF LIPID RAFTS, Src and INTEGRIN Î±vÎ²3 IN REGULATING CANCER CELL METASTASIS
The role of lipid rafts in signal transduction cannot be overemphasized. The lipid raft signalling hypothesis proposes that these microdomains spatially organize signalling molecules at the membrane, perhaps in complexes, to promote kinetically favourable interactions that are necessary for signal transduction37. Alternatively, lipid raft microdomains might inhibit interactions by separating signalling molecules, thereby dampening signalling responses37. Simons and Toomre, 2000 reported that Lipid rafts containing a given set of proteins can change their size and composition in response to intra- or extracellular stimuli thus favoring specific protein-protein interactions, resulting in the activation of signalling cascades27. Several lipid-raft dependent signalling pathways that were discussed in their review included: immunoglobulin E signalling; T-cell antigen receptor signalling; glial-cell-derived neurotrophic factor signalling; Ras signalling and Hedgehog signalling27. Ras signalling pathway can turn certain genes that are involve in cell growth and division, meanwhile, hedgehog signalling gives cells information that need to make the embryo develop properly. Thus, mutation in any of these signalling pathways can consequently lead to the formation of malignant cells. These signalling pathways being able to function in the lipid rafts indicates that the lipid rafts in one way or the other can modulate cancer cells metastasis. Furthermore, membrane rafts can be polarized, formation of membrane rafts at both the leading and rear edge of the cells, by actomyosin contraction and actin polymerization to regulate migration in lymphocyte cells 28.
Integrins are recognized to play several significant roles in the metastasis of cancer cells via aiding its attachment to the ECM. During these interactions some integrins expression are increase and others are decreased often triggering myriad of comprehensive pathways leading to the degradation and remodeling of the ECM, cytoskeletal organization, cell adhesion and survival. Integrins weakly bind to its ligands compared to other cell surface receptors30. This provided the notion that cells quickly attach to and detach from the surrounding substrate, providing a molecular basis for cell motility and invasion. Integrins signalling between the cells and the environment can be group into inside-out signalling and outside-in signalling. Inside-out signalling refers to regulating of integrin function from within the cell, changing it from a passive, weak binding state into an active, adhesive state and thus altering the interaction of these receptors within the extracellular environment31. Meanwhile, outside-in signalling involves the binding of extracellular ligand, enhancing separation of the cytoplasmic domains, as result allowing their interaction with cytoskeletal and signal transduction molecules. Outside-in signalling can control behavior, proliferation, cell polarity, cell growth and cell migration. Integrin expression and affinity profiles are altered in cells to modulate their adhesive capacity and intracellular signalling, thus enabling the cells to assume a more invasive and migratory phenotype29. Î±vÎ²3 positive cells have been discussed to enhance survival of cells enabling them withstand a form of apoptosis called integrin mediated death38. Manes et al., 2003 in their research work have reported that integrin Î²3 can modulate migration in LNCaP prostate cancer cells by regulating the cdc2 mRNA and protein levels39. Thus the role of Î±vÎ²3 in tumor metastasis cannot be overemphasized, it is not surprising that the first Phase III clinical trial with an integrin antagonist was against Î±vÎ²3 and Î±vÎ²5 in glioblastoma38. This indicates the significant role of Î±vÎ²3 in cancer metastasis and the need for more research geared towards unraveling this integrin heterodimer in other cancer cells.
The Src kinases plays several crucial roles in cell signalling, based on this several preclinical trials of a variety of inhibitors have been investigated. Mayer and Krop, 2010 reported that in some cancer cases like prostate cancer the clinical trials are even in the third phase. Among the notable inhibitors in clinical trials include; dasatinib, bosutinib saracatinib, AZM475271, XL999. Notwithstanding the authors were quick to note that due to the several redundancies in the cellular pathways more research is needed to bring to light the clinical relevance of Src inhibition in solid tumors5. The Src family of protein tyrosine kinases (SFKs) plays key roles in regulating signal transduction by a myriad set of cell surface receptors within different cellular environments. Its roles can both be felt at the extracellular matrix and also within the cytoplasm. Furthermore, its function cut across different types of cells ranging from immune cells as in B- and T- cell development, signalling and activation; mediating signalling from cell surface receptors in hematopoietic cells; upregulating the activity of N-methylD-aspartate (NMDA) receptors and other ion channels in the central nervous system thus underlying both physiological (as in learning and memory) - and pathological (as in pain and epilepsy) - plasticity40. Src is also expressed in some cancers including colon, mammary, pancreatic and so on. Parsons and Parsons, 2004 also enumerated various other primary cellular processes of Src, among which include; promoting signalling from growth factor receptor to regulating the turnover of certain cell surface receptors; modulating actin cytoskeleton rearrangements and promoting cell motility and survival; targeting focal adhesion kinase (FAK), paxillin and p130Cas thus regulating integrin-mediated signalling pathways; regulating cell-cell adhesion through its p120-catenin substrate that modulates CDHII40. In support of this, Mitra and Schlaepfer, 2006 have given detail accounts of how FAK and Src can cooperate to bring about cancer cell metastasis. The authors explained further that this regulation depends partially on a linkage to integrin41. In addition, Meng et al., 2009 research revealed that inhibiting Src showed a decrease in phosphorylation of FAK at its Tyr397, Tyr861 and Tyr 925 ultimately leading to the decrease in both invasion and migration of A549 lung cancer cell42.
The Crosstalk Between Lipid Rafts, Src and Integrin Î±vÎ²3
Different proteins may colocalise with the lipid rafts via numerous ways. Some proteins attached to the exoplasmic leaï¬‚et of the bilayer by their GPI anchors, others bind to the cytoplasmic leaï¬‚et by acyl tails and another group of proteins also associate through their transmembrane domains4. Integrin can associate with the lipid rafts through their transmembrane domain; meanwhile Src requires dual acylation that is palmitoylation and myristoylation to enable it to associate with the rafts. Nevertheless, some other SFK members like Lyn, Yes and Fyn is considered to reside in lipid rafts of the plasma membrane, through their N-terminal sequences after myristoylation and palmitoylation. Colocalization of integrins with lipid rafts to enhance the trafficking and internalization of molecules have been extensively reported. A research by Huang et al., 2006 demonstrated that disruption of lipid rafts by emodin hampered colocalization of integrin Î²1 and focal adhesion complexes leading to inhibition of tumor cell adhesion7. The role of lipid rafts in integrin-dependent adhesion has been reported in mouse embryonic neural precursor cells43. In another article, the authors showed that Bordetella adenylate cyclase toxin first binds the integrin Î²2 promoting the relocation of the toxin-receptor complex into specific lipid rafts and thus supporting its translocation directly into the cytoplasmic compartment of cells44. Guan, 2004 observed that lipid rafts are commandeered by integrins to target Rho and Rac GTPases to specific plasma membrane domains and to couple them to their downstream effectors molecule. The author also reported that in migrating cells, local activation of integrin signalling via FAK and perhaps other mediators prevents internalization of plasma membrane lipid rafts45.
Integrins lack kinase activities and for that matter they recruit and activate kinases such as FAK and Src required for their functions in cell migration and survival. Putnam et al., 2009 research have revealed that Src inconjuction with PKCÎ± and PKCÎ´ were involved in Î±vÎ²3 integrin-mediated invasiness in C8161.9 and M14 melanoma cells. However, the authors revealed that the Î±vÎ²3 dependent invasion of M14 was due to overxpression of Src and PKCÎ± or PKCÎ´46. It is generally believed that unligated integrins on adherent cells can induce a form of apoptosis called integrin-mediated death (IMD). A recent review has discussed that Î±vÎ²3-Src signalling module could facilitate a FAK-independent survival pathway of these IMD-resistant tumor cells38. It has also been discussed that integrin clustering could concomitantly result in co-clustering of associated Src molecules, thereby facilitating transphosphorylation and activation6 . They also cautioned that the direct binding of Src to integrin Î² tails presently seems unique to platelet-speciï¬c integrins6. However, a later research using HBL100 epithelial cell line has showed that Î²3 cytoplasmic domain is a critical regulator of c-Src-mediated oncogenic signalling47. Thus the crosstalk between Î±vÎ²3 and Src is very essential in tumor metastasis. Whether Src kinases, which has only myristoylated motif, can localize within the lipid rafts, since dual acylation is crucial for attachment to lipid rafts has over the years been a controversial issue. Recently, Seong et al., 2009 using KRas-Src- and Lyn-Src -FRET biosensor analysis revealed that the Src activity is differentially regulated at different compartments of the plasma membrane, mediated by different sets of cytoskeletal components. Based on their work, the authors proposed that there are two different sets of Src kinases at the plasma membrane48. One set of Src kinases is pre-stored outside of lipid rafts on plasma membrane at rest state and can be rapidly activated upon stimulation 48. Whereas, the other set of Src kinases is located in endosome-like structures around nucleus at rest state, which can be translocated to lipid rafts through actin filaments upon stimulation and become activated48. Membrane rafts are classified into basically two distinct groups, including: the flask shaped protein-based membrane domains as caveolae and flat shaped lipid-based domains as lipid rafts49. The authors also reviewed that in most instances the caveolae and lipid rafts exist as separate structures; however, they can associate with each other49. Caveolin-1(Cav1) is a major important component of the flask shaped caveolae membrane rafts. There are several studies that have reported that Cav1 is a critical regulator of src-dependent tumor cell migration and invasion. A research has revealed that lipid rafts and Cav1 are very important in MDA-MB-231 cells invadopodia-mediated ECM degradation52 . Thus, if Src activities can be regulated by Cav1, then there could be a possibility of lipid rafts also regulating Src activity since caveolae and lipid rafts at one point in time can associate with each other. Given that cancer cells must undergo adhesion and migration to enhance its metastasis, it follows that the cells must have specialized signalling mechanisms to regulate these processes. This study proposes that Src can also regulate integrin Î±vÎ²3 function by recruiting the later into the lipid rafts to bring about metastasis in A375 melanoma cancer cells. Clearly, understanding the fundamental mechanisms that governs how Src regulates integrin Î±vÎ²3 signalling within the lipid rafts to enhance cancer cell metastasis would go a long way to create therapeutic interventions that may have a considerable impact on the treatment of cancer.