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-Synuclein and the progression of cancer
* Department of Biochemistry and
Department of Pharmacology and Therapeutics, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates; and
Biomedical Sciences Unit, Department of Biological Sciences, Lancaster University, Lancaster, UK
1Correspondence: Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates, Al Ain, PO BOX 17666, United Arab Emirates. E-mail: o.elagnaf{at}uaeu.ac.ae
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ABSTRACT |
|---|
-, ß-, and 
-synucleins (-syn). They are a natively unfolded group of proteins that share sequence homologies and structural properties (1, 2). So far, the biological functions of the synucleins are still unclear, but their involvement in neurodegenerative diseases and cancer may provide insights into the pathological processes that result from these two groups of debilitating diseases, and present the possibility to use them as potential targets for early diagnosis and treatment.
Recently, elevated levels of -syn proteins have been detected in various types of cancer, especially in advanced stages of the disease. Furthermore, studies to date indicate that overexpression of -syn compromises normal mitotic checkpoint controls, resulting in multinucleation as well as faster cell growth. -Syn has also been shown to promote invasion and metastasis in in vitro assays as well as in animal models. Overexpression of -syn also interferes with drug-induced apoptotic responses. These observations raise questions about the involvement of -syn in the process of tumorigenesis and metastasis, and efforts have already been made to use -syn as a marker for assessing breast cancer progression (3). This review will discuss the involvement of -syn in cancer progression, metastasis and its potential as a marker.—Ahmad, M., Attoub, S., Singh, M. N., Martin, F. L., El-Agnaf, O. M. A. -Synuclein and the progression of cancer.
Key Words: biomarker • apoptosis • invasion
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INTRODUCTION |
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, 5)
. Their expression seems to be developmentally regulated, with the initial expression of - and ß-syn being observed following synaptic development in primary hippocampal neurons in culture (9)
. -, ß-, and -Synuclein (syn) proteins all share a characteristically conserved N-terminal domain that consists of 5–6 repeats of the KTKEGV consensus sequence in the first 87 residues (6)
. These repeats result in the formation of conserved amphipathic A2-helices also characteristic of apolipoproteins, which mediate reversible binding to phospholipid membranes (7)
. This property supports the role of synucleins in vesicular release at presynaptic nerve terminals (4)
. The interaction with lipid membranes stabilizes the synuclein protein structure from an unfolded to a more folded -helical structure (8)
. However, -, ß-, and - syn differ considerably in their acidic C-terminal domains, which is presumably where their functional differences arise.
The first of the synucleins to be discovered was -syn. It was isolated from the electric ray Torpedo Californica in 1988 by Maroteaux and coworkers, who also coined the term "synuclein," due to the localization of the -syn in presynaptic terminals (10
, 11)
. Its role in the pathogenesis of neurodegenerative diseases was implicated when the hydrophobic sequence of amino acids spanning 61–95 of the -syn protein was discovered to be identical to the non-Aß component (NAC) of amyloid plaques in Alzheimer’s Disease (AD) (12)
. Furthermore, -syn was found to be the major component of cytoplasmic filamentous inclusions found in Lewy bodies (LBs) and Lewy neurites (LNs), the hallmarks of sporadic Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) (13)
. -Syn is also found in glial cytoplasmic inclusions in multiple systems atrophy (MSA) (14)
. This group of neurodegenerative diseases is otherwise known as "synucleinopathies" (15)
.
In 1990 Nakajo et al. identified a 14 kDa phosphoneuroprotein (PNP 14), which was later named ß-syn, due to its high sequence homology to -syn (6)
.
The last of the synucleins to be discovered was -syn. It was first named breast cancer-specific gene 1 (BCSG1) due to its abundant levels in advanced infiltrating breast carcinoma, compared to its almost undetectable levels in normal or benign breast lesions (16)
. On surveying cDNA databases, the BCSG1 and the -syn gene (SNCG) was found to have almost identical gene sequences; due to its high sequence homology with - and ß-syn, it was renamed as a member of the synuclein protein family (17)
.
The human -syn gene (SNCA) is localized on chromosome 4q21.3-q22 (18)
. Alternative splicing of SNCA results in the production of three isoforms of -syn (19)
.The full-length 140 amino acid long isoform, which has the common N-terminal domain found in all synucleins; the central hydrophobic region (NAC), unique to itself, stretches from residues 61–95; and the acidic C-terminal domain. In the brain, its expression has been detected mainly in the presynaptic nerve terminals in the neocortex, hippocampus, striatum, thalamus, and cerebellum cerebellar cortex, substantia nigra, brain stem, and other forebrain structures (6
, 20
21
22
23
24)
. -Syn displays lower expression in the pancreas, retina, optic nerve, heart, and skeletal muscles (25
, 26)
. The shorter form is 112 amino acids long and is missing the residues 103–130; its expression has been observed in both fetal and adult brain. Last of all is the 126 amino acid long -syn which is missing exon 3 (refer to Fig. 1
) (19)
.
|
In addition to the cytosol, the synaptosome and the presynaptic nerve terminals, -syn was found to have a more widespread subcellular localization, including the nucleus (27
28
29)
. The translocation of -syn into the nucleus is brought about by toxic insults to the cell, such as oxidative stress and treatment with paraquat. This promotes interaction of -syn with histones, which may lead to the mechanisms underlying pathophysiology of synucleinopathies (30
, 31)
. In normal and neoplastic Schwann cells, -syn immunoreactivity was found to be distributed in various subcellular localizations, including plasma membrane, ribosomes, rough endoplasmic reticulum, Golgi apparatus, small vesicles, and nuclear outer membrane (32)
.
At high concentrations, wild-type -syn can self-aggregate from solution to form LB-like fibrils and discrete spherical assemblies. However, this process is accelerated in the mutated forms of -syn; studies have associated SNCA mutations with the outcome of familial cases of PD (33
34
35)
. The presence of LBs in dopaminergic neurons in the substantia nigra pars compacta, whose loss is characteristic of PD (36
, 37)
, suggests that the aberrant accumulation of aggregated -syn may be involved in the death of these neurons.
Pathogenic point mutations such as the A53T in familial PD and A30P in familial PD (34
, 38)
and the E46K in familial Parkinsoniam and DLB (35
, 39)
have all been shown to accelerate -syn protein oligomerization (33, 40–42, 43). The A30P mutation disrupts the ability of -syn protein to bind to lipid rafts and synaptic vesicles (45
, 46)
; A53T and A30P mutations has been associated with impaired dopamine storage (47)
; and could promote dopamine-induced oxidative stress in the cytoplasm arising from the toxic accumulation of dopamine in the cytoplasm (44
, 47
, 48)
.
Genetic duplications and triplications of the SNCA locus have also been reported in familial cases of PD, suggesting that increase in gene dosage of SNCA, which concurrently results in an increase in levels of wild-type -syn protein, is also pathogenic. Duplications of SNCA closely resemble idiopathic PD with late-age onset, slow progression and the absence of dementia and cognitive decline (49)
. Alternatively, SNCA triplications, result in early onset PD with faster progression and dementia (50)
.
The post-translational modifications of -syn, such as phosphorylation, oxidation, and nitration, may also be involved in the conversion of soluble monomeric -syn to insoluble -syn aggregates in synucleinopathies (51
52
53
54
55)
. For example, phosphorylation of -syn at position 129 (54)
by casein kinase II is influenced by stress and promotes fibril formation (51
, 56
57
58)
. Phosphorylated -syn as well as -syn nitrated at tyrosine residues interferes with -syn’s ability to interact with phospholipids. Phosphorylated -syn has also been shown to interfere with -syn’s ability to regulate phospholipase D2 activity, and thereby may be affecting -syn’s role in vesicular trafficking and neurotransmitter release (47
, 59)
.
To date, the known functions of -syn include: regulation of synaptic transmission and plasticity (60
, 61)
; dopamine metabolism (62)
; regulation of the vesicular pool in presynaptic terminals of hippocampal neurons (9)
and chaperone-like activity mediated by the C-terminal tail (63
, 64)
. Wild-type -syn seems to possess both neuroprotective and neurotoxic properties, depending on its concentration and expression levels in the neurons (65)
. Wild-type and mutant -syn at micromolar or overexpressed levels promote neuronal death through mitochondrial-dependent apoptosis. This is achieved by the up-regulation of Bcl-2 associated X protein (Bax) expression and the down-regulation of long(L)form Bcl-like (X) protein (Bcl-xL) expression; as well as by interacting with Bcl-xL/Bcl-2-Associated Death Promoter (Bad), a proapoptotic member of Bcl-2 family (63
, 65
, 66)
. In vitro cell models have also demonstrated the effect of overexpressing levels of -syn protein on mitochondrial deficits through oxidative stress (67
, 68)
. On the one hand, the overexpression of both mutant and wild-type -syn disrupts its role in the regulation of dopamine metabolism which has also been shown to result in oxidative stress caused by dopamine toxicity that eventually leads to cell death (37)
.
On the other hand, nanomolar levels of soluble, recombinant -syn protein have been shown to protect neurons from environmental stress such as oxidative stress caused by hydrogen peroxide, serum deprivation, and glutamate toxicity. This is thought to be achieved by acting through the PI3K/Akt signal transduction pathway to increase Bcl-2 family expression levels (65)
.
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ß-SYN |
|---|
-syn and shares a conserved C-terminal domain containing three identically-placed tyrosine residues (6
, 18)
. ß-Syn shares similar subcellular localization (27)
and expression patterns to -syn in the brain. However, apart from weak ß-syn mRNA staining in skeletal muscle its expression in other tissues has not been detected (6)
. Due to the absence of the central 11 hydrophobic amino acid residues (15)
that are thought to contribute to the aggregation of -syn, ß-syn displays less-folded and compact aggregates compared to those formed by - and -syns (69)
. Two ß-syn mutations, V70M, and P123H, have been identified in highly conserved regions of the protein, which may be associated with predisposition to DLB (70)
. ß-Syn shares the chaperone activity of the other synucleins in inhibiting aggregation of thermally denatured alcohol dehydrogenase and chemically denatured insulin while also protecting ocular cells from misfolded and aggregated mutant rhodopsin accumulation, also shared by -syn (64
, 71)
. ß-Syn also assumes a neuroprotective role by inhibiting -syn aggregation and toxicity (69
, 72
73
74
75)
. Transgenic mouse models have demonstrated the neuroprotective activities of ß-syn including the reduction of -syn protein expression and aggregation with accompanying improvements in survival rates and motor dysfunctions (76
, 77)
. These neuroprotective activities of ß-syn have led to the investigation of the potential use of ß-syn peptides as a therapeutic means to prevent the pathogenic activity of aggregated -syn in tissue culture and transgenic mouse models (77
, 78)
. |
-SYN
|
|---|
-syn gene (SNCG) is localized on chromosome 10q23 and encodes a smaller 123 amino acid protein (79)
. The search for genetic mutations on SNCG yielded two linked, naturally occurring polymorphisms that differentiate two transcriptionally active alleles, which suggested overexpression of wild-type -syn protein in cancer might be associated with malignancy (80)
.
The C-terminus of -syn protein diverges completely from the other synuclein members (81)
. This difference is thought to influence the different preferential subcellular localization of -syn from - and ß-syn proteins. The C-terminus of -syn was shown to prefer nuclear localization, while that of the -syn promotes a cytosolic localization (29)
. -Syn protein mainly localizes to the cell bodies and axons of primary sensory neurons, sympathetic neurons and the motor neurons of the peripheral nervous system (81)
. It has recently been discovered that -syn has a dynamic localization, in both neuronal and non-neuronal cell cultures, depending on the activity of the cell. Its presence has been detected in the perinuclear area, but then it has been shown to redistribute to the midbody in late mitosis and can translocate into the nucleus during stress (82)
. -Syn protein has been shown to localize to spindle poles and associate with centrosomal proteins (83)
.
-Syn expression has also been recorded using in situ hybridization in neuronal and non-neuronal tissues. Highest expression of -syn was found in the substantia nigra, hippocampus, thalamus, caudate nucleus, and amygdale. Moderate levels of -syn expression was found in the corpus collosum, heart, skeletal muscle; much lower levels in the kidney, pancreas and lung; no expression has been observed in the liver and placenta (16
, 79)
. -Syn protein expression was also observed in cultured lymphocytes from peripheral blood of healthy individuals (80)
as well as in the retina and optic nerve (25)
. The expression of -syn is thought to be developmentally regulated. This is due to the absence of -syn mRNA and protein levels in embryonic neonatal and forebrain structures, whereas -syn expression is abundant in adult cerebellar cortex, thalamus, hippocampus, and cerebral cortex (80
, 81
, 84)
. This developmental regulation of -syn expression and its involvement in reorganization of the neurofilament network may reflect a role in the formation of the developing nervous system. This hypothesis is supported by the observation that -syn expression coincides with the time when trigeminal ganglions are growing to their targets at embryonic days 10 and 11, after which their expression is then maintained into adulthood (81)
. -Syn may also play a role in the reorganization of neurofilament networks but not the cytoskeletal network. This is suggested due to the drastic reduction in neurofilament-H (NF-H), neurofilament L (NF-L), and neurofilament M (NF-M) on up-regulation of -syn expression (81)
. Both ß- and -Syn proteins share their chaperone abilities as mentioned in the section on ß-syn (64
, 71)
. However, reduction in -syn mRNA and protein levels in the optic nerve and cultured rat astrocytes subjected to elevated hydrostatic pressure, opposes the normal observations of elevated chaperone protein levels during cellular stress (85)
.
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SYNUCLEINS IN NEURODEGENERATIVE DISEASES |
|---|
- and -syn protein levels have been shown to be increased in the brains of sporadic PD and DLB patients, whereas those of ß-syn were shown to be reduced (87)
. However, only -syn and not ß- or -syn proteins, have been shown to be a component of the pathological inclusions found in these PD and DLB brains, such as LBs and LNs (13
, 88)
. The presence of ß- and -syn has been observed in axonal spheroid-like lesions in the hippocampal dentate molecular layer, a novel presynaptic axon pathology in PD brains, but was not observed in the control brain tissues (89)
. The presence of -syn in these axonal inclusions is of interest as -syn is thought to regulate axonal growth during development (81)
. -Syn was also found be elevated in type 2 Gaucher’s disease, which has Parkinsoniam syndrome (90)
. In glaucoma patients, a high level of staining for -syn was observed in a subset of glial cells, presumed to be reactive astrocytes. This was in addition to a cellular redistribution of -syn in the optic nerve of glaucoma patients compared with control patients without glaucoma. All these observations suggested the use of -syn as a potential marker for the disease (85)
.
At the advent of neurodegenerative diseases, the observed increase in ß- and -syn levels could reflect a neuroprotective role to inhibit the aggregation of -syn proteins (69
, 73)
. However, in vivo and in vitro studies conducted on double -syn/-syn null mutant mice suggested that the absence of the -/-syn proteins did not result in a compensatory increase in the levels of ß-syn (91)
. Changes in the expression levels of ß-syn may depend on the expression levels of -syn, in order to protect against the pathogenenic outcome of the aggregated -syn arising from overexpression. That is to say, compensatory increases in the expression of ß-syn levels may only take place in the outcome of overexpressing levels of -syn. On the one hand, this suggests that the fine balance of these synuclein proteins is important in maintaining normal brain function and the disturbance of this balance might lead to the outcome of the synucleinopathies (87)
.
On the other hand, the absence of - and -syn proteins did not seem to have any observational impact on the normal development and survival of sensory neurons in the peripheral nervous system of mice (91
, 92)
. However, the expression of all the synucleins in the growth cones of regenerating neurites suggests that they may be involved in the axonal regeneration following neural damage (93)
.
The function of -syn in the brain is still largely a gray area; its role in neurodegenerative disorders, if any, remains speculative. Studies have tentatively suggested that there could be a certain threshold level after which abnormally high levels of -syn levels becomes toxic to the cell. This comes from the observation that overexpressing -syn neuronal cells promotes degradation of neurofilament proteins by calcium-dependent proteases (94)
. -Syn’s involvement in the reorganization and development of the neurofilament network and high expression levels in the substantia nigra (main target of neuronal degradation in PD), however, has not yet yielded any link to its role in neurodegenerative diseases (79
, 84)
. As mentioned above, the absence of -syn protein did not affect neurons in the nervous system in mice (92)
. This has led to the hypothesis that -syn is dispensable in the development and function of the nervous system (92)
. All of this suggests the presence and function of -syn may be compensated by the other synucleins in its absence and that it may not play a significant role in the brain.
|
SYNUCLEINS IN CANCER |
|---|
-syn, but not - and ß- syns, was seen in 20% of preneoplastic lesions in the ovary, where it showed punctate expression in epithelial inclusion cysts, hyperplastic lesions, and papillary structures (95)
. -Syn was found to be overexpressed in ovarian tumors and in ovarian cancer cell lines in contrast to low and almost undetectable levels of -syn proteins in the surface epithelial cells of normal ovary (17
, 95)
. Initially, BCSG1 was discovered as a differentially expressed gene in human breast cancer and, moreover, was found to be expressed specifically in advanced stages of breast cancer. This is demonstrated by undetectable levels of -syn in normal or benign breast tissues, partial expression in low-grade ductal carcinoma in situ and abnormally high levels in highly infiltrating breast cancer (16)
. This stage-specific expression of -syn suggested that the expression of this protein may be linked to the process of tumorigenesis (16
, 95)
. Furthermore, abnormally high expression of -syn has been associated with a wide range of cancer types, including breast, ovarian, cervical, prostrate, liver, pancreatic, colon, gastric, esophagus, and lung compared to almost undetectable levels in adjacent non-neoplastic tissue (96
, 97)
. In addition to the stage-specific expression of -syn, high expression levels in stage III/IV sometimes involving lymph node invasion, was observed in various cancer types (3
, 16
, 95
96
97
98)
. -Syn expression is also specifically expressed in high-grade glial tumors such as 33% ependymomas, 63% glioblastomas, and 16% myxopapillary ependymomas, which also demonstrates -syn’s potential activity as a tumor progression protein is not just restricted to hormone-dependent carcinomas (99)
. |
-SYN AND CANCER
|
|---|
. To investigate the role of -syn in cancer, we need to investigate whether there are any SNCG mutations associated with the presence of aberrant -syn observed in different cancer types. Sequence studies done on -syn gene revealed no evidence of gene mutations (95)
or gene amplifications in tumor cell lines, which suggests that deregulation at the transcriptional level may be responsible (80)
. |
TRANSCRIPTIONAL DEREGULATION OF SNCG |
|---|
.
The 1 kb 5' flanking region stretches from –1260 to –170. It contains a GC rich binding sequence for Sp1, a transcription factor of the basal transcriptional machinery, located immediately upstream of transcription initiation site (see Fig. 2
). This DNA region provides non-cell-type specific basal transcriptional activity (11)
.
|
Exon 1, stretches from position –169 to +121, contains a CpG island consisting of 15 CpG sites and is the subject of epigenetic transcriptional regulation. Differential DNA methylation of these sites, leads to the cell-type specific -syn expression. Hypomethylation of SNCG has been correlated with abnormally high expression of -syn in diverse cancer types (96
, 101)
. The 15 CpG sites span from –169 to +81 in exon 1, in relation to the translational start codon. Certain breast cancer cell lines (MCF10A, MDA-MB435, MDA-MB468, and MCF-7) that had no or very low -syn expression levels were specifically methylated at sites 2, 5, 7, 10–15. In contrast, exon 1 was unmethylated in breast cancer cell lines (SKBR-3, AU565, BT-20, H3922, MDA-MB231, and T47D) expressing -syn (101)
. However, it seems that different cell types may require a different methylation pattern to silence SNCG expression (101
, 102)
. In ovarian cancer cell lines, complete methylation of all the 15 CpG sites in SNCG is required to switch off -syn expression, whereas partial methylation at the specific CpG sites is sufficient to block SNCG expression in breast cancer cell lines (11
, 96
, 101)
. However, treatment with methyl transferase inhibitors such as 5-aza-2-deoxycytidine, which have been shown to demethylate CpG islands on SNCG, only minimally induced -syn expression. This suggests that there exist other mechanisms that contribute to the overexpression of this gene observed in tumor cells (103)
.
The search for cis-acting regulatory elements of -syn gene expression led to the discovery of activating protein (AP-1) transcription factor consensus binding sites, TGACTCA located across the regulatory region of SNCG. There are two at the 5' flanking region of the gene (from –1194 to –1188 and –488 to –482) and two closely located AP-1 consensus binding sites in intron 1 (103)
(see Fig. 2
). Deletion studies revealed that the two AP-1 sites in the 5' promoter region do not affect the basal promoter activity of SNCG, but the deletion of the two closely located intronic AP1 sites profoundly reduced the basal transcriptional activity of SNCG, resulting in reduced -syn mRNA and protein levels (103)
. AP-1 is a basic leucine zipper transcription factor that forms a dimeric complex consisting of members of JUN and FOS proteins in mammals (104)
. The overexpression of the bovine orthologue of -syn, synoretin, have been shown to up-regulate the erythroblast transformation specific domain containing protein, Elk-1, (which mediates growth factor stimulation through the c-Fos promoter) through the mitogen-activated protein kinase (MAPK) signal transduction pathway (83)
. In breast cancer cells overexpressing -syn, the main components of the AP-1-DNA complex are c-Jun homodimers, with the c-Jun/c-Fos heterodimer constituting a minor component of this complex (11)
. Mutations or deletions in either of these AP-1 motifs lead to a drastic reduction in -syn promoter activity and significantly reduced -syn expression, ultimately resulting in a profound inhibition of cell growth of breast cancer cell lines (11
, 103)
. This supports the role of -syn as a protein involved in tumor progression (103)
. Intron 1 seems to be responsible for the non-cell-type specific expression of -syn. The 5' end of intron 1 includes a regulatory sequence named, repressive element 1 (RE1), spanning + 293 to + 499, responsible for suppressing -syn transcription in the -syn-negative human hepatoma cell line (HepG2) but not human breast cancer cell lines. On the other hand, the 3' end of intron 1, from position + 599 to + 935, contains a positive regulatory sequence that favors cell-type independent expression, localized to AP-1 binding site at + 612 (11)
.
It is still not clear what causes -syn overexpression, but recently it has been observed that hepatic carcinogens aflatoxin B1 and N-nitrosodimethylamine strongly induce SNCG expression through demethylation (105)
. This suggests that the overexpression of -syn observed in some cancer cell types may not be an instigator but rather a marker of cancer progression.
-Syn overexpression may not always be associated with tumor progression. This comes from the observation that -syn mRNA and protein levels are down-regulated in 60% human eosophageal squamous cell carcinoma (hESCC) (106)
. This exception from the general trend seen so far for breast and ovarian cancer tumorigenesis caused by -syn overexpression, indicates a more complex cell specific network of intracellular mechanisms that drive tumor development and progression.
|
ROLE OF -SYN IN TUMOR PROGRESSION
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|---|
, 108)
. These growth factors then bind to their respective cell-surface receptors, which are often overexpressed in cancer types, and amplify the cell’s response to proliferative signal transduction (107)
. Even though -syn does not have any sequence homology to growth factors, and other known oncogenes, its overexpression and involvement in tumor progression has been studied in a range of different cancer cell types. So far, -syn has been shown to be involved during the key steps in the process of tumor progression, as summarized in Table 1
. A more in-depth look at the role played by -syn overexpression on each of these stages is discussed in the proceeding sections.
|
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-SYN IN CELL PROLIFERATION
|
|---|
-syn in breast cancer cell lines led to an increase in cell growth under anchorage dependent and independent conditions (101
, 109
, 110)
. This increase in growth was significantly reduced along with drastic reduction in SNCG expression on treatment with a growth-inhibitory cytokine, oncostatin M (110)
. The specific effect of -syn on the increase in anchorage-independent growth was demonstrated by significant suppression of -syn mRNA using antisense technology, which resulted in the suppression of this growth in human breast cancer cell line, T47D (103)
.
To investigate the intracellular mechanism by which -syn influences tumor progression it would be useful to identify the proteins that interact with it. This was achieved by screening for proteins using the yeast two-hybrid method, which revealed the association of -syn with a mitotic check point protein called BubR1 (111)
. This interaction showed that -syn association with BubR1 leads to a reduction in BubR1 protein by 26S proteasomal degradation but no decrease was observed in the mRNA levels (111)
. Thus, -syn expression leads to over-riding the mitotic arrest allowing cells to continue progression through the cell cycle resulting in aneuploidy, a phenomenon that has been frequently observed in human cancer cell types (111
, 112)
. Furthermore, -syn overexpression suppressed caspase 3 and caspase 9 activity disrupting the nocodazole-induced apoptotic response that would otherwise have resulted from normal BubR1 function (113)
. The interaction of -syn with BubR1 also interferes with BubR1’s association with the microtubule motor protein centromeric protein E (CENP-E), thus disrupting normal mitotic checkpoint signaling (114
, 115)
. This compromise in the normal mitotic checkpoint functions was shown in both estrogen receptor (ER)-positive and ER-negative breast cancer cells lines overexpressing -syn and was evidenced by reduced cell division control protein 2 (Cdc2) kinase activity and phosphorylated histone H3, markers for mitotic arrest. On the other hand, the oncogenic role of -syn on cell proliferation was shown to be strongly dependent on ER- expression. Studies have revealed a novel role of -syn, which seemed to act as a new member of the heat-shock protein-based multiprotein chaperone complex, by enhancing high ligand-binding affinity of ER-, and its subsequent activation leading to its increased transcriptional activity (116
, 117)
. Further support for the effects of -syn on cell proliferation comes from the ability of the bovine orthologue of -syn, synoretin, to activate MAPK and the downstream Elk1 (83)
. MAPK signaling pathways are thought to induce cell proliferation (144)
, and disruptions in these signaling pathways may also be involved in the aberrant changes that take place in neurodegenerative diseases (85)
.
-Syn protein has been shown to localize to spindle poles and associate with centrosomal proteins (83)
. Aberrant function of the centrosome could cause chromosomal abnormalities, aneuploidy, and formation of multiple spindle poles observed in cancer cells (118
119
120)
. The role of -syn as a centrosomal protein is not clear as yet, and whether its overexpression may cause abnormal functions of the centrosome remains to be explored.
|
HOW IS -SYN LINKED TO CELL INVASION AND METASTASIS?
|
|---|
. This is achieved by the action of soluble, secreted proteases such as the matrix metalloproteinases (MMPs), whose expression and action are carefully regulated at both transcriptional and post-transcriptional levels. The overproduction of MMPs, through AP-1 transactivation, has been associated with promoting cancer cell migration and invasion (122
123
124
, 140)
. One of the mechanisms by which -syn overexpression may be involved in tumor progression and malignancy may come from its strong induction of MMP9, tissue inhibitor of MMPs 1 and 2 (TIMP1, TIMP2) expression, and moderate increase in MMP2 activity and protein levels. Retinoblastoma cell lines overexpressing -syn were shown to have higher MMP9 promoter activity, resulting in higher MMP9 protein levels and activity, which may be mediated by AP-1 (125)
. Surgucheva and his co-workers have hypothesized that overexpression of -syn in cancer may up-regulate MAPK pathways, which in turn can phosphorylate and activate AP-1 leading to increase in MMP gene expression. More studies need to be carried out to explore the relationship between AP1 activation and the role of -syn in cancer to resolve these questions (125)
.
To determine if -syn was a mere by-product or the main instigator of malignant progression of breast cancer, -syn was overexpressed in MDA-MBA-435 breast cancer cells. On overexpression of -syn in these cell lines, an increase in cell motility and invasiveness was observed using in vitro assays (109)
. -Syn was also shown to cause metastasis in nude mice on implanting -syn expressing cells in fat pads of these mice. Immunohistochemical results showed mice given implants of -syn positive cells displayed an increase in tumor growth, and metastasis into axillary lymph nodes and lungs, compared with mice given control implants (-syn-negative cells) (109)
.
|
THE EFFECT OF -SYN OVEREXPRESSION ON CELL SURVIVAL
|
|---|
, 127
, 141)
. Mutations in these death-inducing ligands and their signaling pathways have been implicated in series of non-small cell lung carcinomas with lymph node metastasis (128)
and highly malignant breast cancers (129
130
131)
. Both pathways converge on the activation of a cascade of proteases called caspases that activate downstream signaling pathways, including c-Jun N-terminal kinase 1 (JNK1) to induce apoptosis (132
, 141)
.
Chemotherapeutic drugs and environmental stress signals, such as ultraviolet rays, arsenate, and heat shock kill cancer cells through JNK1 signaling pathway via caspase-3 dependent apoptosis (132
, 133)
. One of the other oncogenic activities of -syn overexpressing cells include an increase in resistance to chemotherapeutic drugs, such as taxol or vinblastine, upon the inhibition of JNK signaling (132)
. Other chemotherapeutic drugs, such as paclitaxel, act by inhibiting extracellular signal-regulated kinases 1 and 2 (ERK1/2). This pathway has been shown to be involved in cell survival and is constitutively active in human ovarian cancer (134)
. Moreover, activated ERK2 has been associated with resistance to cisplatin-induced apoptosis (135)
. Cell lines overexpressing -syn promote the constitutive activation of ERK1/2 while down-regulating JNK1 in response to environmental stress signals, including ultraviolet, arsenate, and heat shock. -Syn overexpression was not able to block etoposide induced apoptosis, as this drug does not act through the JNK signaling pathway (132)
. Taken together all these results indicate that -syn overexpression promotes cancer cell survival and can abrogate the apoptotic action of chemotherapeutic drugs by inhibiting the activation of the two MAPKs.
|
SYNUCLEINS AS POTENTIAL BIOMARKERS |
|---|
-syn to be secreted into medium by cultured neuronal cells in vitro as well as in human lumbar cerebrospinal fluids and blood plasma (136
, 142)
. The presence of -syn in the biological fluid has raised the possibility of using this protein as a biomarker for the early detection and diagnosis of PD (136
, 142)
. The potential for using -syn as a biomarker for early diagnosis of PD is currently being investigated after a novel ELISA was developed to specifically detect oligomeric forms of -syn in CSF (137)
. Studies have already indicated a decreased level of the -syn protein in CSF can be used as a marker for PD (143)
. Another study has shown -syn expression levels are elevated in the peripheral blood mononuclear cells of PD patients (138)
.
So far it has been suggested that aberrant expression of -syn is triggered early in the tumor development process and is playing a role in tumor progression and metastasis. The possibility of using -syn as a biomarker for tumor progression comes from detecting -syn protein levels in the sera of 38% cases of pancreatic adenocarcinomas, while none was detected in those of normal controls (97
, 139)
. Li et al. 2004 have suggested a more sensitive method of detecting -syn in biological fluids using ELISA may reveal a higher percentage of cancer types overexpressing -syn (97)
.
Most recent studies have shown that -syn expression in breast cancer is involved with poor prognosis and has the potential to be used as an independent biomarker for breast cancer progression (3)
. The expression of -syn in a stage-specific manner in breast (16
, 98
, 132)
, ovarian (95)
, pancreatic (97)
and more recently in hepatocellular carcinomas (105)
suggests that it could act as an indicator of malignant progression of cancer. This has been followed up by Wu et al. 2006, who have also found a correlation with -syn expression and lower disease-free survival (3)
. Public serial analysis of gene expression (SAGE) was employed to detect -syn levels secreted into serum and urine samples as a test toward using -syn as a biomarker for pancreatic cancer (97)
. Thus, -syn is proving to be a promising potential biomarker as an indicator of cancer stage and survival to be used in clinical studies, without the need for the application of more invasive methods. Studies have not been carried out, as yet, to explore the mechanisms by which -syn is released from the cell into the vascular system, but it would be interesting to see if the synucleins share the mechanism by which they are released into the blood sera, as was proposed for -syn (136)
.
|
ACKNOWLEDGMENTS |
|---|
Received for publication March 1, 2007. Accepted for publication May 10, 2007.
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-synuclein on dopamine homeostasis in a new human mesencephalic cell line. J. Biol. Chem. 277,38884-38894This article has been cited by other articles:
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