Download Deep Insight Section The tumour suppressor function of the scaffolding protein spinophilin

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The tumour suppressor function of the
scaffolding protein spinophilin
Denis Sarrouilhe, Véronique Ladeveze
Laboratoire de Physiologie Humaine, Faculte de Medecine et Pharmacie, Universite de Poitiers, 6 rue
de la Miletrie, Bat D1, TSA 51115, 86073 Poitiers, Cedex 9, France (DS), Laboratoire de Genetique
Moleculaire de Maladies Rares, Universite de Poitiers, UFR SFA, Pole Biologie Sante, Bat B36, TSA
51106, 86073 Poitiers, Cedex 9, France (VL)
Published in Atlas Database: February 2014
Online updated version : http://AtlasGeneticsOncology.org/Deep/SpinophilinID20133.html
DOI: 10.4267/2042/54041
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence.
© 2014 Atlas of Genetics and Cytogenetics in Oncology and Haematology
Abstract
Spinophilin is a scaffolding protein with modular domains that govern its interaction with a large number of
cellular proteins. The Spinophilin gene locus is localized at chromosome 17q21, a chromosomal region
frequently affected by genomic instability in different human tumours. The scaffolding protein interacts with the
tumour-suppressor ARF which has suggested a role for Spinophilin in cell growth. More recently, in vitro and in
vivo studies demonstrated that Spinophilin is a new tumour suppressor acting via the regulation of pRb. A clear
downregulation of Spinophilin is found in several human cancer types. Moreover, Spinophilin loss is associated
with a poor patient prognosis in carcinoma. Currently, there are controversial findings regarding a functional
relationship between Spinophilin and p53 in cell cycle regulation and in carcinogenesis. Here we present the
available data regarding Spinophilin function as a tumour suppressor.
Actin-Binding proteIN), and the latter was further
identified as Spn (Nakanishi et al., 1997). Spn is
expressed ubiquitously while neurabin 1 is
expressed almost exclusively in neuronal cells. Spn
exhibits the characteristics of scaffolding proteins
with multiple protein interaction domains (Allen et
al., 1997; Sarrouilhe et al., 2006). Scaffolding
proteins link signalling enzymes, substrates and
potential effectors (such as channels, receptors) into
a multiprotein signalling complex that may be
anchored to the cytoskeleton. In the years after this
discovery, the spectrum of Spn partners and
functions has expanded but has remained mostly in
the field of neurobiology (Sarrouilhe et al., 2006).
Spn has been implicated in the pathophysiology of
several central nervous system (CNS) diseases,
among which are Parkinson's disease, schizophrenia
and mood disorders (Law et al., 2004; Brown et al.,
2005). Spn is highly enriched at the synaptic
membrane in dendritic spines, the site of excitatory
neurotransmission and thus may control PP1
functions during synaptic activity (Ouimet et al.,
Key words
CaSR, G protein-coupled receptor, signaling
1- Introduction
Protein phosphatase 1 (PP1) is a widespread
expressed phosphoSerine/phosphoThreonine PP
involved in many cellular processes (Ceulemans
and Bollen, 2004). There are four isoforms of PP1
catalytic subunit (PP1c): PP1α, PP1β, PP1γ1 and
PP1γ2, the latter two arising through alternative
splicing (Sasaki et al., 1990). PP1c can form
complexes with up to 50 regulatory subunits
converting the enzyme into many different forms,
which have distinct substrates specificities,
restricted subcellular locations and diverse
regulations (Cohen, 2002). In late 1990s, a novel
PP1c binding protein that is a potent modulator of
PP1 activity was characterized in rat brain and
named spinophilin (Spn) (Allen et al., 1997). In the
same time, two novel actin filament-binding
proteins were purified from rat brain and named
neurabin 1 and neurabin 2 (NEURal tissue-specific-
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The tumour suppressor function of the scaffolding protein spinophilin
2004). Spn regulates plasticity at the postsynaptic
density (PSD) by targeting PP1c to α-amino-3hydroxy-5-methylisoxazole-4-propionic
acid
(AMPA) and N-methyl-D-aspartic acid (NMDA)
receptors, promoting their down regulation by
dephosphorylation and thus regulating the
efficiency
of
post-synaptic
glutamatergic
neurotransmission. Spn and neurabin1 play
different roles in hippocampal and striatal synaptic
plasticity. Spn is involved in long-term depression
(LTD) but not in long-term potentiation (LTP)
whereas neurabin 1 contributes selectively to LTP
but not LTD (Feng et al., 2000; Allen et al., 2006;
Wu et al., 2008). In the same way, the two
scaffolding proteins form a functional pair of
opposing regulators that reciprocally regulate
signalling intensity by some seven-transmembrane
domain receptors (Wang et al., 2007). Thus, an
emerging notion is that Spn and neurabin 1 may
differentially affect their target proteins and
perform quite distinctive function in cell.
Morphological studies have established that Spn is
enriched at plasma membrane of cells although the
protein is also expressed widely throughout the
cytoplasm (Smith et al., 1999; Richman et al., 2001;
Tsukada et al., 2003). Spn, which is expressed
partly in the nucleus in mammalian cells, interacts
in vitro and in vivo with the tumor-suppressor ARF
(Alternative Reading Frame). Moreover, a role for
Spn in cell growth was suggested, and this effect
was enhanced by the interaction between Spn and
ARF (Vivo et al., 2001). More recent studies
showed that Spn is a new tumour suppressor and
that a clear downregulation of this protein is found
in several cancer types (Carnero, 2012).
Furthermore, Spn loss is associated with poor
patient prognosis in carcinomas (Sarrouilhe, 2014).
This review aims to outline the state of knowledge
regarding Spn function in carcinogenesis.
domains, a PSD95/DLG/zo-1 (PDZ) and three
coiled-coil domains. Figure 2 provides a schematic
diagram of the main Spn structural domains.
In the five species of the Figure 1, the coiled-coil
region has high identity with only one variation
detected in Cricetulus griseus. The PDZ domain,
the pentapeptide motif of PP1c -binding domain
and the sextapeptide allowing the binding
selectivity of PP1c isoforms, present the same
identity. Moreover, the phosphoSer are conserved
except the Ser-177 which is only detected in rat.
Being not detected in mouse (G as in primates),
Ser-177 is not a consequence of the rodent-specific
high substitution rate.
Spn has been isolated from rat brain as a protein
interacting with F-actin (Satoh et al., 1998). Its Factin-binding domain determined to be amino
acids 1-154 is both necessary and sufficient to
mediate actin polymers binding and cross-linking.
Nuclear Magnetic Resonance (NMR) and circular
dichroism (CD) spectroscopy studies showed that
Spn F-actin-binding domain is intrinsically
unstructured and that upon binding to F-actin it
adopts a more ordered structure (a phenomenom
also called folding-upon-binding). Another actin
binding property, namely a F-actin pointed end
capping activity was recently proposed for this
domain (Schüler and Peti, 2007). Spn, PP1c and Factin can form a trimeric complex in vitro.
A receptor-interacting domain, located between
amino acids 151-444, interacts with the third
intracellular loop (3i) of various seven
transmembrane domain receptors (Smith et al.,
1999; Richman et al., 2001) such as the dopamine
D2 receptor (D2R), some subtypes of the αadrenergic (AR) and muscarinic-acetylcholine (mAchR) receptors.
The primary PP1c-binding domain is located
within residues 417-494 of Spn and this domain
contains a pentapeptide motif (R-K-I-H-F) between
amino acids 447 and 451 that is conserved in other
PP1c regulatory subunits. A domain C-terminal to
this canonical PP1-binding motif, located within
amino acids 464 and 470, is essential for PP1
isoform selectivity in vitro and for selective
targeting in cells (Carmody et al., 2008).
Recently, the 3-dimentional structure of the
PP1/Spn holoenzyme was determined. Spn is an
unstructured protein in its unbound state that
undergoes a folding transition upon interaction with
PP1c into a single, stable conformation. The
scaffolding protein binds to PP1c and blocks some
potential substrate binding sites without altering its
active site, then didacting substrate specificity of
the enzyme (Ragusa et al., 2010). A further study
showed that the PP1/Spn holoenzyme is dynamic in
solution.
2- Spinophilin structure
The primate (homo sapiens and Callithrix jacchus)
Spn proteins contain 815 amino acids whereas the
rodent Spn (rattus norvegicus and mus musculus)
have 817amino acids. These sequences are very
similar, with few amino acids substitutions
compared to the human sequence in C-terminus but
the N-terminus is more variable even if the
variability is weak (Figure 1). Consequently, few
differences are observed when we compared these
sequences to the human one: the rat and human Spn
proteins share 96% sequence identity (Allen et al.,
1997; Vivo et al., 2001). In Cricetulus griseus, the
sequence is shorter than the others: 631amino acids.
Gene analysis and biochemical approaches have
contributed to define in Spn a number of distinct
modular domains. This 130 kDa protein contains
one F-actin-, a receptor- and a PP1c- binding
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The tumour suppressor function of the scaffolding protein spinophilin
Sarrouilhe D, Ladeveze V
Figure 1. Alignment of amino acid sequences of spinophilin in different species. Blast and Align programs via UniProt site
were used.
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Figure 2. Schematic drawing of spinophilin structure. The canonical protein phosphatase 1-binding domain is located within
amino acids 447 and 451 in spinophilin.
signalling protein (like RGS8), guanine nucleotide
exchange factors (like kalirin 7), membrane
receptors [like the α-ARs, m-AChRs, D2R, δ- and
µ-opioid receptors (OR) and cholecystokinin (CCK)
receptors], and other proteins like ions channels
[The transient receptor potential canonical (TRPC),
the type 2 ryanodine receptor (RYR2)], TGN38 and
ARF.
Shortly after the cloning of Spn as a novel PP1cbinding protein, another laboratory cloned this
protein based on its ability to bind to F-actin (Satoh
et al., 1998).
Recombinant Spn and neurabin 1 interacted with
each other when co-expressed in cells. On the other
hand, recombinant Spn was shown to form
homodimers, trimers or tetramers by interaction
between coiled-coil domains. Spn homomeric
complexes are thought to contribute to its actincross-linking activity (Satoh et al., 1998).
Doublecortin (DCX) is a microtubule-associated
protein that can induce microtubule polymerization
and
stabilize
microtubules
filaments.
Immunoprecipitation experiments with brain
extracts showed that Spn and DCX interact
incultured cells (Tsukada et al., 2003).
In vitro assays showed that DCX also binds to and
bundles F-actin, suggesting that the protein crosslinks microtubules and F-actin.
The distribution of DCX between the two
cytoskeletons can be regulated by Spn and by
phosphorylation of DCX and it was proposed that
Spn could localize and enhance the binding of
phosphorylated DCX to F-actin (Tsukada et al.,
2005).
Several studies have shown that Spn preferentially
binds to PP1γ1 and PP1α isoforms in brain extracts
(MacMillan et al., 1999; Terry-Lorenzo et al., 2002;
Carmody et al., 2004).
GST-Spn fusion proteins containing the PP1cbinding domain potently inhibit PP1 enzymatic
activity in vitro (Allen et al., 1997; Colbran et al.,
2003).
However, it was recently shown that instead of
inhibiting PP1c directly, Spn regulated enzymatic
activity by directing its substrate specificity
(Ragusa et al., 2010).
Spn can associate with the tyrosine phosphatase
SHP-1 and the complex modulates platelet
The complex adopts a significant more extended
conformation in solution than in the crystal
structure. This is the result of a flexible linker
(ramino acids 490-494) between the PP1c-binding
and the PDZ domains. The four residue flexibility
is likely important for Spn biological role (Ragusa
et al., 2011).
Spn also contains a single consensus sequence in
PDZ, amino acids 494-585 (Allen et al., 1997). The
structure of the Spn PDZ domain has been recently
solved by NMR spectroscopy. The PDZ domain
directly binds to carboxy-terminal peptides derived
from glutamatergic AMPA and NMDA receptors
(Kelker et al., 2007).
Sequence analysis predicted that the carboxyterminal region of Spn (amino acids 664-814)
forms 3 coiled-coil domains. Neurabins were
observed as multimeric forms in vitro and in vivo.
Spn and neurabin 1 homo- and hetero-dimerize via
their
carboxy-terminal
coiled-coil
domains
(MacMillan et al., 1999; Oliver et al., 2002).
Consensus sequences for phosphorylation by
several protein kinases (PK), including cAMPdependent PK (PKA), Ca2+/calmodulin-dependent
PK II (CaMKII), cyclin-dependent PK5 (Cdk5),
extracellular-signal regulated PK (ERK) and protein
tyrosine kinases were observed in Spn. Two major
sites of phosphorylation for PKA (Ser-177 not
conserved in human, and Ser-94) and two others
sites for CaMKII phosphorylation (Ser-100 and
Ser-116) were located within and near the F-actinbinding domain of Spn. The protein is
phosphorylated in intact cells by PKA at Ser-94 and
Ser-177 and by CaMKII at Ser-100 (Hsieh-Wilson
et al., 2003; Grossman et al., 2004). Moreover,
neurabins can be phosphorylated in vitro and in
intact cells by Cdk5 on Ser-17 and ERK2 (MAPK1)
on Ser-15 and Ser-205, phosphoSer-17 being
abundant in neuronal cells (Futter et al., 2005).
Several potential tyrosine phosphorylation sites lie
within the coiled-coil regions, within a region
adjacent to the PDZ domain and within the
receptor-interacting domain.
3- The Spinophilin interactome
Spn interactome includes cytoskeletal molecules
(F-actin, doublecortin, neurabin 1, Spn), enzymes
(like PP1 and CaMKII), regulator of G-protein
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The tumour suppressor function of the scaffolding protein spinophilin
nervous system. Spn was identified with other
dendritic spines proteins as a protein partner of
TRPC5 and TRPC6 channels (Goel et al., 2005). In
cardiomyocytes, Spn targets PP1 to RYR2 via
binding to a leucine zipper (LZ) motif of RYR2 and
a LZ motif on Spn (amino acids 300-634) causing
dephosphorylation and modulation of the channel
activity (Marx et al., 2001).
TGN38 is an integral membrane protein that
constitutively cycles between the trans-Golgi
network (TGN) and plasma membrane via
endosomal intermediates. TGN38 directly interacts
with the coiled-coil region of Spn, preferentially
with the dimerized proteins (Stephens and Banting,
1999). Spn has been shown to interact with the
nuclear protein ARF in mammalian cells. The
amino acids sequence 605-726, of the coiled-coil
region of Spn, seems to be involved and an intact
ARF N-terminal region (amino acids 1-65) is
necessary for this interaction (Vivo et al., 2001).
activation by sequestering RGS10 and RGS18. The
sequence surrounding the phosphorylation site
Y398 in Spn fits a consensus ITIM sequence
(I/V/L/SxY(p)xx(I/V/L) and forms a binding site
for SHP1 (Ma et al., 2012). p70S6K is a mitogenactivated PK that regulates cell survival and
growth. p70S6K interaction with neurabin 1
(Burnett et al., 1998) and Spn was demonstrated
(Allen and Greengard, unpublished observation).
The interaction implicates the PDZ domain of
neurabins and the carboxyl-terminal five amino
acids of the PK. CaMKII directly and indirectly
associates with N- and C-terminal domains of Spn.
Thus, Spn can target CaMKII to F-actin as well as
target PP1 to CaMKII (Baucum et al., 2012).
Regulator of G-protein signalling (RGS) proteins
play a crucial role in the shutting off process of Gprotein-mediated responses (Ishii and Kurachi,
2003). Spn binds to different members of the RGS
family (Wang et al., 2005; Wang et al., 2007). For
example, Spn binds to through the 391-545 amino
acids of the scaffolding protein and the 6-9 amino
acids of the N-terminus of RGS8 (Fujii et al.,
2008).
Guanine nucleotide exchange factors (GEF)
activate small G protein through the exchange of
bound GDP for GTP. Several GEF were shown to
interact with Spn. For example, Spn, through its
carboxy-terminus containing the PDZ and coiledcoil domains interacts with kalirin-7, the neuronal
GEF for Rac1 (Penzes et al., 2001).
Spn interacts with some receptors that belong to the
superfamily of GPCRs, mainly in the CNS. Using
the 3i loop of the D2R, Spn has been identified as a
protein that specifically associates with the receptor
in rat hippocampal (Smith et al., 1999). The 3i
loops of α2A-AR, α2B-AR, and α2C-AR subtypes
interact also with Spn (Richman et al., 2001). More
recently, it has been shown that the α1B-AR interacts
with Spn in vitro (Wang et al., 2005). In the
cerebellum, Spn can bind to the M1-m-AChR
using the receptor binding domain of the
scaffolding protein (Fujii et al., 2008). Spn can also
interact with the M2- and M3-m-AchRs but the
binding ability to the M3-m-AChR seems to be
weaker than those to the M1- and M2-m-AChR
(Wang et al., 2007; Kurogi et al., 2009). Moreover,
Spn binds to the 3i loop of CCKA and CCKB
receptors (Wang et al., 2007). The receptor binding
domain of Spn also associates with the 3i loop and
a conserved region of the C-terminal tails of δ- and
µ-OR (Fourla et al., 2012). Spn also interacts with
the ionotropic NMDA and AMPA-type glutamate
receptors. PDZ domain directly binds to GluR2-,
GluR3- (AMPA receptor) and NR1C2'-, NR2A/Band NR2C/D- (NMDA receptor) derived peptides
(Kelker et al., 2007).
TRPC ion channels are Ca2+ /cation selective
channels that are highly expressed in the central
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4- Spinophilin as a tumour
suppressor
The Spn gene locus is located on chromosome 17 at
position 17q21.33, a cytogenetic area frequently
associated with microsatellite instability and loss of
heterozygosity (LOH) observed in different human
tumours. This region contains a relatively high
density of known (such as BRCA1), putative as well
as several yet-unidentified candidate tumour
suppressor genes located distal to BRCA1 locus.
Thus, several studies in breast and ovarian
carcinomas have suggested the presence of an
unknown tumour suppressor gene in the area that
includes the Spn locus. However, despite these
preliminary genetic correlations, no in-depth
analysis of the role of Spn as a tumour suppressor
has been made.
The Amancio Carnero laboratory from the Instituto
de Biomedicine de Sevilla, in Spain, have
addressed this possibility in vitro and in vivo, in
three articles published in 2011. In the first study,
immunohistochemical analysis of 35 human lung
tumours at different stages and of different
histopathological grades showed that Spn protein is
absent in 20% and reduced in another 37% of
tumours, compared to normal lung tissue (MolinaPinelo et al., 2011). The loss of Spn expression
correlated with a less differentiated phenotype,
higher grade and poor prognosis. Lower or null
levels of Spn also correlated with nuclear
accumulation of p53, and so to mutated p53 or loss
of its wild-type activity. Moreover, loss of Spn
increased the tumourigenic properties of p53
deleted- or p53 mutated-lung tumour cells. The data
of this study showed that Spn down-regulation in
lung tumours contributes to carcinogenesis in the
absence of p53. There are several mechanisms that
might contribute to Spn down-regulation in
695
The tumour suppressor function of the scaffolding protein spinophilin
regulates the expression of p14 (ARF) (Liu et al.,
2012). Some members of the family of e2F
transcription factors are also involved in cell cycle
regulation; in particular E2F1 which expressions
increase induces augmentation of ARF which can
bind MDM2 and stabilize p53. In p53 (- / -) MEF,
reduced levels of Spn enhanced tumorigenic
potential of the cells. Indeed, inhibition of e2F by
Rb being lifted, this results in cell proliferation no
longer controlled by p53. Moreover, the absence of
Spn contributes to genetic alterations during MEF
immortalization, particularly p53 mutations. These
results extend the observations made by the authors
using a Spn-null mice model (Ferrer et al., 2011b).
In summary, the results suggested that Spn is a new
tumour suppressor acting via the regulation of pRb
and which function is revealed in the absence of a
functional p53 (Sarrouilhe and Ladeveze, 2012).
This is, therefore, suggestive of partially redundant
functions in their tumour suppression properties
(Santamaría and Malumbres, 2011). The results
also suggest that the specific outcome can be
context-dependent. Spn loss may be beneficial by
potentiating p53 in response to acute stress, and in
contrast it can be deleterious under sustained
mitogenic stress (Palmero, 2011). This feature is
reminiscent of NIAM (Nucleolar Interaction of
ARF and MDM2 protein) which acts through the
same partners p53 and ARF (Tompkins et al.,
2007).
Another Spn-interacting molecule is DCX, an actinbinding and microtubule-binding protein that seems
to be a tumour suppressor of glioma. When DCX is
ectopically expressed into the DCX-deficient U87
glioma cells, there is a marked suppression of the
transformed phenotype. The cells manifest a
reduced rate of growth in vitro and are arrested in
the G2 phase of the cell cycle. Moreover, DCXtransfected U87 glioma cells do not generate
tumours in immunocompromised nude rats. In
DCX-transfected U87 cells, phosphorylated DCX
binds specifically to Spn and this interaction
inhibits proliferation and anchorage-independent
growth in glioma cells. In contrast, DCX-mediated
growth repression is lost in glioma cells treated
with siRNA to Spn and in HEK 293 (human
embryonic kidney) Spn null cell line (Santra et al.,
2006). DCX, Spn and PP1c were found in the same
protein complex from mouse brain extracts
(Shmueli et al., 2006).
DCX-mediated growth arrest in glioma cells may
be through inactivation of PP1 activity by
Spn/DCX interaction in the cytosol. Inhibition of
PP1 activity is involved in two mechanistic links of
reduction
of
glioma
tumour-associated
progressions: firstly, catastrophe in mitotic
microtubule spindle that blocks mitosis; secondly,
depolymerization of actin that inhibits glioma cell
invasion (Santra et al., 2009).
tumours, including miRNAs overexpression.
miRNA106*, targeting Spn, are overexpressed in a
small subset of patients with decreased Spn levels.
Overexpression of miRNA106* significantly
increased the tumorigenic properties of lung tumour
cells. The results suggested that miRNA106*
overexpression found in a subset of lung tumours
might contribute to tumorigenesis through Spn
down-regulation in the absence of p53. In a second
study, tumour suppression by Spn was explored in
in vivo model using genetically modified mice
(Ferrer et al., 2011b). Spn-null (-/-) mice displayed
decreased survival, increased the number of
premalignant lesions in tissues such as the
mammary ducts and early appearance of
spontaneous tumours, such as lymphoma, when
compared to WT littermates. In another series of
experiments, the presence of mutant p53 activity
(p53R172H) in the mammary glands was evaluated
on a Spn heterozygous (+/-) or homozygous (-/-)
background in mice. An increased number of
premalignant lesions and of mammary carcinomas
were observed in Spn heterozygous (+/-) or
homozygous (-/-) mice when compared to WT
littermates. The results confirmed the functional
relationship between Spn and p53 in tumorigenicity
and showed that Spn loss contributes to tumour
progression rather than the tumour initiation. In a
third study using mouse embryonic fibroblasts
(MEFs), it was suggested that Spn acts as a tumour
suppressor by the regulation of the stability of
PP1cα, thereby regulating its activity on pRb (the
phosphorylated form of the Retinoblastoma
protein). This function of PP1cα has been
associated with the growth arrest response; the
hypophosphorylated form of Rb protein being the
most abundant when cells are delayed in their
growth (Ceulemans and Bollen, 2004). The ectopic
overexpression of Spn in immortalized MEF greatly
reduced tumour cell growth. Moreover, the absence
of Spn (Spn(-/-) MEF) down-regulated PP1α
activity resulting in a high level of pRb (Ferrer et
al., 2011a). High level of proproliferative
phosphorylated Rb leads to e2F activation, a
compensatory ARF transcription, and consequently
p53 activation. As they regulate the cell cycle, p53
and ARF are both tumour suppressors, which are
themselves regulated by MDM2 (Mouse double
minute 2) protein shuttle between the nucleus and
cytoplasm (Kamijo et al., 1998; Pomerantz et al.,
1998). Moreover, Sherr et al. (2005) suggested for
the first time a p53-independent pathway via the
ARF sumoylation. Ha et al. (2007) described ARF
as a melanoma tumour suppressor by inducing p53independent senescence. Moreover, Du et al. (2011)
demonstrated the functional roles of ERK and p21
for ARF in p53-independent tumour suppression.
Furthermore, in a p53-independent pathway, the
over-expression of wild-type c-myc obviously up-
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Figure 3. Cellular cycle regulation by spinophilin. A. In normal cells, the presence of nuclear p53 and Spn proteins regulates
cell cycle. The binding of PP1ca to Spn allows dephosphorylation of pRb, which inhibits E2F1 and thus the proliferation.
Furthermore both tumour suppressors (p53 and ARF) regulate the cell cycle. The nucleolar ARF is also a partner of Spn, and
regulates the cell cycle via Mdm2 and E2F1. B. In the case of colorectal carcinomas, Spn play a role in regulation of cell cycle
via a p53/ARF independent pathway. One hypothesis suggested by the team of Amancio Carnero is that the Ras/Raf pathway
could be implicated (Estevez-Garcia et al., 2013). This cytoplasmic pathway could be regulated by cytoplasmic Spn. K-Ras:
GTPase, oncogene; B-Raf: serine/threonine protein kinase, proto-oncogene; Mek: tyrosine/threonine kinase (Mapk kinase);
Mapk: mitogen-activated protein kinase.
diagnosed after the 10-year follow-up in 85.2%
cases with Spn low expression and 60.9% with Spn
high expression. Death occurred in 76.5% cases
with Spn low expression and in 56.5% cases with
Spn high expression. Overall, low Spn expression is
a factor for poor prognosis in hepatocellular
carcinoma. In vitro experiments (human hepatoma
cell line HepG2) and in vivo observations (Ki67positive tumour cells) showed that reduced Spn
expression significantly correlated with a higher
proliferation of liver cancer cells (Aigelsreiter et al.,
2013).
In the second study, the role of Spn was explored in
colorectal carcinoma, in which a number of
chromosomal regions are altered (Fearon, 2011).
Among them, the 17q21 is lost in a high percentage
of this carcinoma (Garcia-Patiño et al., 1998).
Quantitative RT-PCR analysis showed that
approximately 25% of colorectal carcinoma
tumours had a greater than 50% decrease in Spn
mRNA levels compared with normal colonic tissue.
A tissue array of human colorectal carcinomas was
generated to confirm this result by exploring the
presence of Spn protein. 70% of colorectal
carcinomas displayed high Spn levels (similar to
the values observed in normal tissue), 20% showed
intermediate levels and 10% showed no expression
of Spn. Moreover, Spn down-regulation correlated
with a more aggressive histologic phenotype
(higher Ki67-positive tumour cells) and was
associated with faster relapse and poorer survival in
patients with advanced stages of colorectal
carcinoma. The data also suggested that Spn loss
induced a chemoresistance in patients with
advanced stages of colorectal carcinoma that had
received adjuvant fluoropyrimidine chemotherapy
Moreover, double transfection with DCX and Spn
reduced self-renewal in brain tumour stem cells via
incomplete cell cycle endomitosis (Santra et al.,
2011). But, is there relevance for Spn as a
prognostic marker in patients with cancer? Spn is
absent in 20% and reduced in another 37% of
human lung tumors (Molina-Pinelo et al., 2011).
A further analysis of Spn in human tumours shows
that Spn mRNA is lost in a percentage of renal
carcinomas and lung adenocarcinomas. A clear
down-regulation of Spn was found in tumoral
samples of the CNS (oligodendrogliomas,
anaplastic astrocytomas, glioblastomas) when
compared to normal nervous samples. Furthermore,
lower levels of Spn mRNA correlate with higher
grade of ovarian carcinoma and chronic
myelogenous leukemia (Carnero, 2012). Two
articles published in spring 2013 associated Spn
loss with poor patient prognosis in patients with
carcinoma
(Sarrouilhe,
2014).
The
17q
chromosomal region is commonly impaired in
hepatocellular carcinoma (Furge et al., 2005). In the
first study, complete loss of Spn immunoreactivity
was found in 42.3% hepatocellular carcinoma and
reduced levels were found in additional 35.6%
cases. Quantitative RT-PCR analysis confirmed in
70% cases a significant reduced Spn mRNA
expression in tumour tissue compared with the
corresponding non-neoplastic tissue. miRNA106*,
targeting Spn in lung tumours, could not be
detected in any of the hepatocellular carcinoma
samples. Moreover, no correlations could be found
for the number of Spn-positive tumour cells and
p53 or ARF staining. These results suggested a
p53-independent tumorigenic role of Spn in
hepatocellular carcinoma. Disease recurrence was
Atlas Genet Cytogenet Oncol Haematol. 2014; 18(9)
697
The tumour suppressor function of the scaffolding protein spinophilin
Sabatini DM, Snyder SH. Neurabin is a synaptic protein
linking p70 S6 kinase and the neuronal cytoskeleton. Proc
Natl Acad Sci U S A. 1998 Jul 7;95(14):8351-6
following surgical resection. Therefore, the
identification of the levels of Spn in advanced
stages of colorectal biopsies has prognostic and
predictive value and might contribute to select
patients who could or could not benefit from
current chemotherapy. In vitro and in vivo
experiments showed no functional relationship
between Spn levels and the presence or absence of
mutated p53 in colon cancer. The authors proposed
that this correlation is dependent on the molecular
context of the tumour cell (Estevez-Garcia et al.,
2013).
5- Discussion and perspectives
We are still only at the early stage in unravelling
the function of Spn in cell cycle regulation. Overall,
the different studies on the tumour suppressor
function of Spn show two pathways of cell cycle
regulation by Spn. The first model is a pathway
dependent of p53 and ARF.
This pathway was previously described in several
articles where Spn interacts with different partners
localized in the nucleus (Figure 3A). The second is
a pathway independent of both molecules. As Spn
is ubiquitously expressed in the cell, the first model
highlights the nuclear localization of Spn and its
interaction with other nuclear proteins.
The second model, more hypothetical, underlines
the possibility that Spn could interact with
cytoplasmic partners. The studies made on
colorectal carcinomas show that Spn could play a
role in a pathway independent of p53/ARF. One
hypothesis is that the Ras/Raf pathway and more
precisely K-Ras/B-Raf is implicated. This pathway,
via Mek (tyrosine/threonine kinase) and Mapk
(mitogen activated protein kinase) induces
transcription factors and proliferation survical
(Figure 3B).
Further studies are needed to elucidate the
underlying mechanisms linking Spn to carcinomas
and expand the prognostic and predictive value of
the Spn expression level to other types of cancer.
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700