1. health and fitness
2. disease
3. cancer

UNIVERSITA’ DEGLI STUDI DELLA TUSCIA
DI VITERBO
DIPARTIMENTO DI AGROBIOLOGIA E AGROCHIMICA
CORSO DI DOTTORATO DI RICERCA IN
“GENETICA E BIOLOGIA CELLULARE”
XXIII CICLO
Role of Lamin A/C in neuroblastoma
differentiation: therapeutic implications
BIO/11
Coordinatore: Prof. Giorgio PRANTERA
Tutors: Dr. Igea D‟AGNANO
Dr. Armando FELSANI
Dottorando:
Giovanna MARESCA
To my family
INDEX
ABSTRACT ......................................................................................................................................... 1
1
INTRODUCTION ........................................................................................................................ 3
1.1
NEUROBLASTOMA ........................................................................................................... 3
1.1.1
Neuroblastoma as a sympathetic nervous system-derived tumor .................................. 4
1.1.2
Neuroblastoma cell lines as “in vitro” differentiation model ........................................ 7
1.1.3
Retinoids-induced neuroblastoma cell differentiation ................................................... 9
1.2
NUCLEUS AND NUCLEAR LAMINA ............................................................................ 10
1.2.1
Lamins .......................................................................................................................... 11
1.2.2
Lamins during development and differentiation processes.......................................... 14
1.2.3
Lamins and regulation of the cellular mechanical properties ...................................... 16
1.2.4
Role of lamins in chromatin organization and gene expression .................................. 17
1.2.5
Role of lamins in DNA replication and cell proliferation ............................................ 18
1.2.6
Laminopathies .............................................................................................................. 19
1.2.7
Lamins and cancer ....................................................................................................... 22
2
AIMS .......................................................................................................................................... 23
3
MATERIALS AND METHODS ............................................................................................... 24
3.1
Cell lines maintenance, differentiation and treatments ....................................................... 24
3.2
Generation of recombinant viruses and lentiviral infection of cells ................................... 24
3.3
Western Blot analysis .......................................................................................................... 26
3.4
Immunofluorescence microscopy........................................................................................ 26
3.5
Total RNA preparation ........................................................................................................ 26
3.6
Real‐time RT‐PCR analysis ................................................................................................ 27
3.7
Whole genome expression profiling.................................................................................... 27
3.8
Plating Efficiency Assay ..................................................................................................... 30
3.9
In vitro Cell Migration Assay.............................................................................................. 30
3.10
Zymography of Gelatinolytic Activity ................................................................................ 30
3.11
P-Glycoprotein immunostaining ......................................................................................... 31
3.12
Calcein/AM Retention Assay .............................................................................................. 31
4
RESULTS ................................................................................................................................... 32
4.1
Silencing of LMNA gene inhibits RA-induced differentiation in SHSY5Y cell line .......... 32
4.2
Gene expression profiling of SHSY5Y CTR and miRL cells reveals an impairment of
differentiation ................................................................................................................................. 35
4.3
The impairment of cell differentiation in LMNA silenced SHSY5Y cells is associated with
increased tumor progression........................................................................................................... 38
4.4
Low levels of Lamin A/C expression are associated with increased resistance to
chemotherapeutic agents ................................................................................................................ 41
5
DISCUSSION ............................................................................................................................. 43
6
BIBLIOGRAPHY ...................................................................................................................... 46
APPENDIX ........................................................................................................................................ 58
ABSTRACT
Neuroblastoma is one of the most aggressive solid tumors in childhood. The degree of its
differentiation can influence patient outcome since high differentiated tumors have been correlated
to favourable prognosis. Most neuroblastoma cells maintain the ability to differentiate in vitro in the
presence of various agents, rendering them a good model to study the differentiation potential of
these tumors.
Nuclear lamins are type V intermediate-filament proteins that form a scaffoldlike meshwork
underlying the inner nuclear membrane and have many different and not completely known
functions. Among lamins, the A-type ones are expressed in differentiated tissues and are involved in
the differentiation processes during the embryonal development. Their expression is reduced or
absent in different human malignancies even though their role in the tumorigenesis is not yet
characterized.
Our aim was to investigate the role of Lamin A/C, belonging to A-type lamins, in the
differentiation and tumorigenesis of neuroblastoma cells. We have also studied whether Lamin A/C
could represent a marker of drug sensitivity, which could contribute to develop therapeutic
strategies based on the molecular profile of the individual tumor.
As cellular model we employed a neuroblastoma cell line able to differentiate in vitro and
showing high expression levels of Lamin A/C, the SHSY5Y cell line. As differentiating stimulus
we used the all-trans retinoic acid (RA), the most effective compound which has been shown to
induce differentiation in neuroblastoma cells and that is employed as biological agent in clinics to
improve patients‟ survival.
Silencing of Lamin A/C blocked the differentiation processes activated by the RA in SHSY5Y
cells, thus preventing the formation of neurites and inhibiting the expression of the tyrosine
hydroxylase differentiation marker. The genome-wide gene-expression profiling of SHSY5Y cells
silenced for LMNA gene (miRL cells), in the presence or not of the differentiating stimulus
confirmed that miRL cells have down-regulated those genes necessary for the differentiation
program. As well an increase of tumor progression related genes shifted the SHSY5Y cells
phenotype towards a more aggressive one, with a higher resistance elicited versus some
chemoterapic agents, thus demonstrating that Lamin A/C could represent a good marker of
neuroblastoma tumor progression and of response to antitumoral therapeutics.
1
SOMMARIO
Il neuroblastoma è uno dei tumori solidi infantili più aggressivi. Il suo grado di
differenziamento può influenzare lo sviluppo della malattia e l‟esito clinico poiché più il tumore
risulta differenziato più la prognosi è favorevole. La maggior parte delle cellule di neuroblastoma
mantiene la capacità di differenziare in vitro in presenza di diversi stimoli e ciò le rende un buon
modello per studiare il potenziale differenziativo di tali tumori.
Le lamine nucleari sono proteine che appartengono alla classe dei filamenti intermedi di tipo V.
Formano una struttura reticolare al di sotto della membrana nucleare interna e svolgono diverse
funzioni, alcune delle quali non sono state ancora del tutto chiarite. Tra le lamine, quelle di tipo A
sono espresse nei tessuti differenziati e sono coinvolte nei processi differenziativi che avvengono
durante lo sviluppo embrionale. La loro espressione risulta ridotta o assente in molti tumori umani,
tuttavia non è stato ancora caratterizzato un loro ruolo nei processi di tumorigenesi.
Il nostro obiettivo è stato quello di indagare il ruolo della Lamina A/C, appartenente alle lamine
di tipo A, nel differenziamento e nella tumorigenesi delle cellule di neuroblastoma. Abbiamo inoltre
verificato la possibilità che la Lamina A/C sia un marcatore di sensibilità ai farmaci chemioterapici,
in quanto ciò consentirebbe di sviluppare strategie terapeutiche personalizzate sulla base del profilo
molecolare del singolo tumore.
Come modello cellulare abbiamo utilizzato la linea di neuroblastoma SHSY5Y, che esprime ad
alti livelli la Lamina A/C ed è in grado di differenziare in vitro. Abbiamo scelto quale stimolo
differenziativo l‟acido retinoico (RA), poiché è il composto più efficace nell‟indurre il
differenziamento delle cellule di neuroblastoma ed è inoltre utilizzato in clinica per aumentare la
sopravvivenza dei pazienti affetti da neuroblastoma.
Il silenziamento della Lamina A/C nella linea SHSY5Y ha causato un blocco dei processi
differenziativi attivati da RA, in quanto risultano inibite sia la formazione dei neuriti che
l‟espressione del marcatore tirosina idrossilasi. Lo studio del profilo di espressione genica delle
cellule SHSY5Y silenzate per il gene LMNA (cellule miRL), in presenza o meno dello stimolo
differenziativo, ha confermato una repressione di diversi geni necessari al differenziamento. Le
cellule miRL mostrano inoltre un incremento nell‟espressione di geni relati alla progressione
tumorale che si accompagna ad un fenotipo più aggressivo nonché ad un‟aumentata resistenza ad
alcuni farmaci chemioterapici. L‟espressione della Lamina A/C nel neuroblastoma potrebbe dunque
essere un buon marcatore di progressione tumorale e di risposta alla terapia farmacologica.
2
1
INTRODUCTION
1.1 NEUROBLASTOMA
Neuroblastoma is an embryonic tumor of the autonomic nervous system and is the most
common extracranial solid cancer in childhood. As can be expected with a disease of developing
tissues, neuroblastoma generally occurs in very young children: close to 50 percent of
neuroblastoma cases occur in children younger than two years old .
The clinical presentation of neuroblastoma is extremely variable, ranging from highly
aggressive phenotypes to benign tumors with a high propensity for spontaneous regression (Brodeur,
2003). Three broad risk categories (low, intermediate, and high risk) have been proposed on the
basis of analysis of age at diagnosis, histology category, grade of tumor differentiation, DNA ploidy,
and copy-number status at the MYCN oncogene locus at chromosome 11q. In particular the MYCN
oncogene is target of high-level amplifications at chromosome band 2p24 observed in about 20% of
neuroblastoma and since such amplification has been found to profoundly affect the patients‟
clinical outcome, it is routinely used as prognostic biomarker and for treatment stratification
(Seeger et al., 1985).
Treatment strategies employed in neuroblastoma patients depends on the risk group they belong
to. Patients with localized neuroblastoma (low risk) have excellent event-free survival rates with
surgical excision of tumor only and an overall survival rate higher than 95%. Intermediate risk
group patients include children younger than 18 months with essentially non-MYCN–amplified
tumors. They are subjected to surgery and chemotherapy (drugs employed are cyclophosphamide,
doxorubicin, carboplatin and etoposide). Treatment of high-risk group patients includes multi-agent
chemotherapy, surgery, and radiotherapy, followed by consolidation therapy with high-dose
chemotherapy and peripheral blood stem cell rescue. Control of minimal residual disease with
biological agents has also been shown to improve survival. The most effective compound is 13-cis retinoic acid, which has been shown to induce differentiation in neuroblastoma cells (Matthay et al.,
1999).
The high degree clinical heterogeneity of neuroblastoma reflects the complexity of genetic and
genomic events associated with the development and progression of this disease. Inherited genetic
variants and mutations that initiate tumorigenesis have been identified in neuroblastoma and
multiple somatically acquired genomic alterations have been described that are relevant to disease
progression (Capasso and Diskin, 2010). Familial neuroblastoma is reported in only about 1% of
patients and is caused by a very rare germline mutation in the anaplastic lymphoma kinase (ALK)
3
oncogene (Mosse et al., 2008). These mutations result in a constitutive activation of the kinase and a
premalignant state. Children with either sporadic or familial neuroblastoma usually have loss-offunction mutations in the homeobox gene PHOX2B. Thus, genetic analysis of ALK and PHOX2B
mutations should be considered every time a patient has family history of neuroblastoma (Mosse et
al., 2004). Nevertheless, additional familial genes may still be discovered. A genome-wide
association study of neuroblastoma is currently under investigation, with the support of Children‟s
Oncology Group. To date, the study has shown that a relative common copy-number variation at
1q21 chromosome is associated with the neuroblastoma and that alleles with common singlenucleotide-polymorphism variations within the putative genes FLJ22536 and BARD1 are
significantly enriched in neuroblastoma patients compared with control groups (Capasso et al., 2009;
Maris et al., 2008).
1.1.1 Neuroblastoma as a sympathetic nervous system-derived tumor
The human nervous system consists of the Central Nervous System (CNS), comprising the
brain and the spinal cord, and the Peripheral Nervous System (PNS), which links the CNS with the
body‟s sense receptors, muscles, and glands. The PNS is divided in two components: the somatic or
skeletal nervous system, which controls voluntary movement, and the autonomic nervous system,
which regulates inner organ function via the sympathetic, parasympathetic or enteric ganglia.
The human nervous system originates from the neural plate, an embryonic structure evolving
from the ectodermal germ layer during the third week of gestation. During the process of
neurulation, the neural plate invaginates ventrally and closes in order to form the neural tube, which
will give rise to the CNS. During this closure, neural crest cells originate at the interface between
the closing neural tube and the dorsal ectoderm (LaBonne and Bronner-Fraser, 1999). The neural
tube is initially composed of a single layer of cells. However, as development proceeds and
extensive cell division occurs, the neural tube becomes multilayered, with precursor cells dividing
in the medial portion of the neural tube adjacent to the central cavity, which will give rise to
ventricles. The portion of the neural tube adjacent to the ventricles becomes the Ventricular Zone
(VZ) and contains neural stem cells and dividing progenitors (Greene and Copp, 2009). These stem
cell populations are capable of self-renewing through symmetric cell divisions or can differentiate
through asymmetric cell divisions.
4
Fig. 1.1 Nervous system development (cross-section): invagination of the dorsal ectoderm, closure of the neural tube,
neural crest formation at the interface of the closing neural folds and dorsal ectoderm, and migration of the pluripotent
crest cells (human time frame).
The neural tube expands in the head of the embryo to form the brain and in the trunk to form
the spinal cord. The PNS derives from the neural crest and the ectodermal placodes. Neural crest
cells delaminate from the neural tube and migrate extensively throughout the embryo to generate,
differentiate, and populate numerous organs. The migrating neural crest cells are largely guided
along distinct pathways by specialized adhesion molecules in the extracellular matrix or by
5
molecules on the surfaces of cells in the embryonic periphery. The movement through a varying
cellular environment has important effects on their differentiation. In fact, during their migration
specific growth factors induce neural crest cells to differentiate into distinct phenotypes including
the neurons and glia of the sensory and visceral motor (autonomic) ganglia, the neurosecretory cells
of the adrenal gland, and the neurons of the enteric nervous system. They also contribute to variety
of non-neural structures such as melanocytes, cartilage, dentine, and bone. NSCs can persist in adult
organism as undifferentiated immature populations, and can be recruited to replace dead or
damaged cells following injury or during the course of normal development and aging (Anderson,
1997).
Fig. 1.2 The differentiation of neural crest cells. The establishment of each precursor type relies on signals provided by
one of several specific peptide hormones.
6
Extensive parallel characterization of marker genes expressed in human Sympathetic Nervous
System (SNS) during embryonic and fetal development and in human neuroblastoma, reveals that
these tumors have immature neuronal characteristics, thus indicating their derivation from
precursors cells of the sympathetic lineage (Hoehner et al., 1998).
Recently, it has been proposed that mutations in specific neural stem cells or early neural
progenitors can give rise to a small population (1-2% of the total) which is capable of tumor
initiation. As described in the stem cell hypothesis of cancer initiation, neoplasms may be viewed as
a heterogeneous population of cells formed by cancer cells and cancer stem cells (CSCs) (Bjerkvig
et al., 2005). As normal stem cells, CSCs are capable of self-renewing and differentiation within the
tumor. It has been shown that CSCs can naturally survive antitumoral treatments and could be
responsible for tumor re-growth and metastasis. This resistance can be attributed not only to their
quiescent nature but also to an elevated expression of anti-apoptotic proteins and drug efflux pumps
belonging to superfamily of ABC transporters (Hirschmann-Jax et al., 2004). As a consequence,
more effective chemotherapy could be achieved by specifically targeting also these cells. Although
the very low representativeness of CSCs in tumors they have been identified in several brain tumors,
including neuroblastoma, as well as in established cancer cell lines (Singh et al., 2004) (Walton et
al., 2004). The neuronal CSCs express several surface markers peculiar of stem cells such as the
CD133, CD34, ABCG2, and nestin (Mahller et al., 2009).
1.1.2 Neuroblastoma cell lines as “in vitro” differentiation model
The degree of neuroblastoma differentiation has been related with the patient outcome, high
differentiation stage correlating to favorable prognosis (Hedborg et al., 1995). Nevertheless, the
mechanisms governing neuroblastoma cell differentiation are only partially understood. In such a
rare malignancy as neuroblastoma, access to fresh tumor material is limited and in vitro studies
based on primary tumor explants are difficult to perform. The possibility to use long term cultures
derived from neuroblastoma tumors allowed to study the peculiar properties of such tumors and to
understand the propensity of neuroblastoma cells to differentiate in vitro and in vivo. As previously
stated, neuroblastoma cells are very heterogeneous; usually they are undifferentiated, round and
small with scant cytoplasm, but most of them retain the capacity to differentiate in vitro following
an adequate stimulus (Edsjo et al., 2007).
7
Many established neuroblastoma cell lines contain at least 3 distinct morphological variants
(Walton et al., 2004) (Ciccarone et al., 1989). The most common variant are the sympathoadrenal
neuroblasts (N-type), which grow as poorly attached aggregates of small, rounded cells with short
neuritic processes. The S-type cells are instead large and flattened cells that attach strongly to the
substrate. They resemble Schwannian, glial or melanocytic progenitor cells and they are nonmalignant. The third cell type is termed I because its phenotype is intermediate between the N and S
type. I-type cells are small, flattened and moderately adherent, and have the potential to
differentiate into N- or S-type cells (Ross et al., 1994).
The ability of many neuroblastoma cell lines to differentiate in the presence of various agents
(e.g. retinoids and phorbolesters such as 12-O-tetradecanoyl phorbol-13-acetate) and growth factors
(NGF, BDNF) has led to the use of neuroblastoma cell lines as model systems to study tumor cell
maturation in general and human neuronal differentiation in particular (Edsjo et al., 2007).
One of the hallmarks of neuroblastoma differentiation in culture is the ability of the cells to
form neurites, a property which is peculiar of normal neurons. During development, neurons
assembled into functional networks by growing out axons and dendrites (collectively called neurites)
which connect neurons each other through particular junctions, named synapses (or synaptic
terminals), and allow the interneuronal transmission of electrical or chemical signals.
Fig. 1.3 General Structure of a Neuron. Each neuron has three basic parts: cell body (soma), one or more dendrites, and
a single axon with a synaptic terminal.
8
Additional markers evidenced in neuroblastoma differentiated cells are the expression of
neurofilaments; the synthesis of neurotransmitter biosynthetic enzymes; the expression of opioid,
muscarinic and neurotrophin receptors; the presence of dense core granules, presumed sites of
catecholamine storage; the expression of neuron specific enolase (NSE). When differentiated,
neuroblastoma cells present either an adrenergic phenotype producing relatively high levels of
tyrosine hydroxylase (TH) and dopamine-ß-hydroxylase (DBH), or a cholinergic phenotype
showing a high activity of choline aceltyltransferase (ACHE) (Hill and Robertson, 1997) (Handler
et al., 2000).
1.1.3 Retinoids-induced neuroblastoma cell differentiation
Among the most widely used agents that induce terminal differentiation reducing cell growth in
selected neuroblastoma cell lines are retinoids. (Sidell, 1982).
The retinoids (all-trans-retinoic acid or ATRA, 13-cis retinoic acid and 9-cis retinoic acid) are a
class of polyisoprenoid lipids which include vitamin A (retinol) and its natural and synthetic
analogs. Retinoids are compounds with multiple functions. They are involved in the control of cell
proliferation, cell differentiation, and embryonic development. Vitamin A is indispensable for
embryonic pattern formation, for visual function, and the differentiation of epithelial tissues.
The signaling pathway of retinoids involves specific nuclear receptors that regulate gene
expression: the retinoic acid receptors (RAR α, β, γ) and the retinoic X receptor (RXR α, β, γ).
These non-steroid nuclear hormone receptors bind their retinoid ligands, target to retinoic
responsive elements on DNA and up- or down-regulate transcription. Neuroblastoma cells
constitutively express RARα, RARγ, RARβ and RXRs although the levels of RARβ are reduced
compared to RARα and RARγ (Li et al., 1994). Neuroblastoma treatment with ATRA besides
inhibiting cell growth results in a decreased anchorage-independent growth, and in the expression of
some neuronal differentiation markers such as extensive neuritic processes, ultrastructurally and
electrophysiologically similar to those of normal neurons; increased neuron specific enolase activity;
slight accumulation of norepinephrine; up-regulation of GAP43, coding for the growth associated
protein 43, a protein important in axonal growth. (Pahlman et al., 1984). Moreover, decrease in a
number of proto-oncogenes (MYCN, MYB, HRAS), increase in the TrkB and RET receptors
expression and activity, were shown to precede the morphological differentiation (Thiele et al.,
1988).
9
As a therapeutic tool, retinoids, such as 13-cis-retinoic acid (isotretinoin), have been shown
promising in reducing the risk of recurrence after high-dose chemotherapy and stem cell transplant.
Recently, potentially more effective retinoids, such as fenretinide, are now being studied in clinical
trials (Villablanca et al., 2006).
Fig. 1.4 Scheme of the intracellular pathways involving retinoids (CRBP: cellular retinol binding protein; CRABP:
cellular retinoic acid binding protein; RARE: retinoic acid response element; RBP: retinol binding protein; RoDH:
retinol dehydrogenase.)
1.2 NUCLEUS AND NUCLEAR LAMINA
In eukaryotic cells, the nucleus is a large membrane-bound organelle which houses the genetic
material. The nucleus is separated from the cytoplasm by a nuclear membrane known as the nuclear
envelope (NE) that consists of two parallel cellular membranes, an inner and an outer membrane,
separated by 10 to 50 nanometers. The outer nuclear membrane (ONM) is continuous with the
membrane of the rough endoplasmic reticulum (RER) and is studded with small openings called
nuclear pore complexes (NPCs), which allow the movement of selected molecules in and out of the
nucleus. In fact, the NE acts as a selective barrier controlling the traffic of macromolecules,
including proteins and RNAs. The space between the membranes is called the perinuclear space and
is continuous with the RER lumen. The inner nuclear membrane (INM) is lined by a complex
meshwork of intermediate filament proteins forming the Nuclear Lamina (NL) that structurally
10
supports the NE and largely determines the overall shape of the interphase nucleus (Gerace and
Burke, 1988).
Fig. 1.5 Schematic representation of nucleus structures
1.2.1 Lamins
The major components of the NL are type V intermediate filament (IF) proteins, the nuclear
lamins. Like all proteins, they are synthesized in the cytoplasm and then transported into the
nucleus, where they are assembled before being incorporated into the existing network of NL (Aebi
et al., 1986).
Lamins are divided into A and B types based on sequence homologies. In mammals, two major
A-type lamins (Lamin A and C) and two major B-type lamins (Lamin B1 and B2) have been
characterized. Minor isoforms such as the Lamin AΔ10 (Machiels et al., 1996), the germ cellspecific lamins C2 (Furukawa et al., 1994) and B3 (Furukawa and Hotta, 1993), have been also
identified. While B-type lamins are encoded by different genes, Lamins A and C are derived from
one gene, LMNA (located on chromosome 1q21.2-q21.3), by alternative splicing ((Broers et al.,
11
2006); (Schumacher et al., 2006); (Verstraeten et al., 2007)). These two latter lamins are produced
in roughly equal amounts.
Little is known about the regulation of lamins gene expression. Slight informations are
available on the regulation of LMNA gene expression. LMNA promoter presents different regulatory
motifs among which a retinoic acid-responsive element (Okumura et al., 2000), binding sites for
various transcription factors such as c-Jun, c-Fos and Sp1/3 (Okumura et al., 2004) or
transcriptional coactivator such as CREB-binding protein (Janaki and Parnaik, 2006). Within the
first intron of LMNA gene there are also binding sites for two transcription factors, the hepatocyte
nuclear factor-3β and the retinoic X receptor β (Arora et al., 2004).
The lamins consist of an amino (N)-terminal globular domain, a central α-helical coiled-coil
rod domain and a globular carboxy (C)-terminal tail domain. Like the components of other
intermediate filaments, the lamin alpha-helical domain is used by two monomers to coil around
each other, forming a dimer structure called “coiled coil”. Two of these dimer structures then join
side by side, in an antiparallel arrangement, to form a tetramer called “protofilament”. Eight of
these protofilaments are laterally combined and twisted to form the characteristic 10 nm
intermediate filament structure. These filaments can be assembled or disassembled in a dynamic
manner. Although A- and B-type lamins interact each other in vitro (Sasse et al., 1998), little is
known about their composition and structure in the cells within the lamina. Lamins contain a
structural motif similar to a type of immunoglobulin fold (Ig-fold) within their C-terminal domain
(Dhe-Paganon et al., 2002). Unique among the other intermediate filaments, lamins contain also a
nuclear localization signal (NLS) and, with the exceptions of lamins C and C2, a C-terminal CAAX
motif (where C is cysteine, A is an aliphatic amino acid and X is any amino acid), which allows to
extensive post-translational modification of these proteins.
Fig. 1.6 Schematic structure of mature lamin A and lamin C polypeptide chains. The lamin structure consists of a short
amino terminal head domain, a central α-helical rod domain (red), and the carboxy-terminal domain containing the NLS
and the Ig-fold (blue; the nine b-strands of the Ig-fold motif are depicted).
12
Lamins A, B1, and B2 are initially expressed as pre-lamins that require extensive posttranslational modifications of their carboxy-terminal –CAAX box to become mature lamins
(Rusinol and Sinensky, 2006). The maturation process starts with farnesylation at the terminal
cysteine site and is followed by cleavage of the last three amino acid residues of the CAAX motif
and methylation of the carboxy-terminal cysteine. While the maturation of B-type lamins is
terminated at this step, resulting in permanent farnesylation and carboxymethylation, an additional
15 amino acids are removed from the carboxyl terminus of farnesylated/carboxymethylated
prelamin A (Corrigan et al., 2005). The last cleavage probably takes place during or after
incorporation of this molecule into the nuclear lamina. Lamin C, which is 74 residues shorter than
mature Lamin A, does not possess a –CAAX box and therefore is not farnesylated or otherwise
modified.
Besides to farnesylation and carboxymethylation, lamins are also posttranslationally modified
by phosphorylation (Ottaviano and Gerace, 1985), sumoylation (Zhang and Sarge, 2008), ADPribosylation (Adolph, 1987), and possibly by glycosylation (Ferraro et al., 1989). At the onset of
mitosis, the phosphorylation of lamins at specific sites by cyclin-dependent kinase 1 (Cdk1) and
protein kinase C (PKC) is required to drive disassembly of the lamina (Dessev et al., 1988; Goss et
al., 1994). Subsequently, dephosphorylation of the lamins by protein phosphatase1a is required for
lamin/lamina assembly during the telophase/early G1 transition (Thompson et al., 1997).
13
Fig. 1.7 Posttranslational processing of the carboxyl terminus of prelamins A, B1, and B2. Processing takes place in a
series of steps: (1) addition of a farnesyl group to the cysteine residue of the –CAAX box of pre-lamin A, prelamin B1
and prelamin B2 by a farnesyltransferase; (2) removal of the last three residues (2AAX) by an AAX endopeptidase; (3)
methylation of the terminal carboxylic acid group (2COOH) by a carboxyl methyltransferase; (4) removal of the
carboxyl terminal 15 amino acids of lamin Awith the farnesyl attached by the metalloprotease Zmpste24/FACE1. This
last proteolysis step does not occur on B-type lamins and therefore they remain farnesylated.
1.2.2 Lamins during development and differentiation processes
Studies of the early development of Xenopus have shown that the individual types of lamins are
differentially expressed in early development and that the changes in the nuclear lamina
composition correlate with major programs in cell differentiation (Benavente et al., 1985). Similar
changes in lamins expression have been reported in early developing Drosophila (Riemer et al.,
1995) and chicken embryos (Lehner et al., 1987).
In mammals, although at least one B-type lamin is expressed in all cells throughout
development, the expression of A-type lamins is regulated during development (Schatten et al.,
1985). In mice, B-type lamins are expressed during early development, while acquisition of the
Lamin A/C expression varies according to developmental stage of tissue differentiation (Stewart
and Burke, 1987) (Rober et al., 1989). This has been confirmed in several studies using human and
14
mouse cultured cells (Lin and Worman, 1997). More recently it has been shown that B-type lamins,
but not A-type ones, are expressed in undifferentiated mouse and human embryonic stem (ES) cells.
The regulated expression of A- and B-type lamins is also evident during differentiation of stem cells
in culture. In fact, during the in vitro differentiation process, human ES cells appear to express
Lamin A/C before a complete down-regulation of the pluripotency marker Oct-4, suggesting that
Lamin A/C expression is an early indicator of ES cell differentiation (Constantinescu et al., 2006).
Differential expression of A- and B-type lamins has also been shown during neurogenesis in the
adult rat brain (Takamori et al., 2007).
Comprehensions into the role of lamins during development come from the analysis of
mutations in the B-type Drosophila lamin Dm0 that cause lethality at different embryonic or late
pupal stages (Osouda et al., 2005). Further insights into the requirement for a specific lamin isoform
during development have been obtained from knocking out either LMNA or LMNB1 in mice. The
Lamin A/C-deficient animals develop normally until birth, but have severe postnatal growth
retardation and develop muscular dystrophy (Sullivan et al., 1999). By contrast, mice that are null
for Lamin A but express Lamin C and the B-type lamins appear to be healthy. On the other hand,
Lamin B1-deficient mice die at birth and have defective lungs and bones (Vergnes et al., 2004).
The developmental regulation of Lamin A/C expression has led various laboratories to
hypothesize that these proteins play a role in differentiation. This has been recognized in muscle
and adipocyte differentiation processes. More specifically, in the case of muscle, during myoblast
differentiation there are changes in Lamin A/C organization strictly dependent from the tumor
suppressor pRb (Muralikrishna et al., 2001). Expression of a mutant form of Lamin A in C2C12
myoblasts induces elevated levels of hyperphosphorylated pRb, leading to an inhibition of the
differentiation of myoblasts into myotubes (Favreau et al., 2004). In addition, in vitro myogenesis
is impaired in LMNA-null skeletal myocytes
(Frock et al., 2006). Otherwise, several LMNA
mutations have been linked to the development of lipodystrophies. In fact, it is known that the
adipocyte differentiation factor, sterol response element binding protein 1 (SREBP1), interacts with
the C terminus of Lamin A/C and that this interaction is impaired to various degrees by mutations in
Lamin A (Lloyd et al., 2002; Hubner et al., 2006), thus contributing to the dysregulation of
adipocyte differentiation.
15
1.2.3 Lamins and regulation of the cellular mechanical properties
The highest and well-established function of nuclear lamins is to provide shape and mechanical
stability to the nucleus (Goldman et al., 2005). In support of this, the nuclei of LMNA knockdown
fibroblasts display increased deformability and impaired viability under mechanical strain (Houben
et al., 2007). Increased nuclear deformability is also observed in human ES cells lacking A-type
lamins (Pajerowski et al., 2007). Moreover, cells either deficient in lamins or expressing mutant
lamin proteins often contain deformed nuclei (Muchir et al., 2003; Bechert et al., 2003; Vaughan et
al., 2001).
Furthermore, several evidences support a role for the lamins in maintaining the mechanical
properties of the entire cell by forming not only a complex network in the nucleus but also a bridge
between the nucleus and the cellular membrane via the cytoskeleton. Indeed, Lamin A/C deficiency
causes impaired mechanotransduction and decreased mechanical stiffness (Lammerding and Lee,
2005). These alterations may be explained by modifications in connections between the
nucleoskeleton and the cytoskeleton. Specifically, the connection between the cytoplasm and
nucleoplasm might be mediated by the interaction between integral proteins of INM (the Sun
proteins Sun1 and Sun2) and of ONM (the nesprins nesprin-1, nesprin-2, and nesprin-3α) in the
luminal space (Padmakumar et al., 2005; Ketema et al., 2007). In the nucleoplasm Sun proteins
interact with Lamin A (Haque et al., 2006), while in the cytoplasm, nesprins are thought to bind to
actin (Warren et al., 2005) and possibly microtubules (Wiche, 1998). This assembly is referred to as
the LINC complex (for LInker of Nucleoskeleton and Cytoskeleton) and establishes a physical
connection between the nucleoskeleton and the cytoskeleton (Crisp et al., 2006).
16
Fig. 1.8 A model for the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex. Nuclear components, including
lamins, bind to the inner nuclear membrane SUN domain proteins, which in turn bind to the KASH domain of the actinassociated giant nesprins on the outer nuclear membrane. The LINC complex establishes a physical connection between
the nucleoskeleton and the cytoskeleton.
1.2.4 Role of lamins in chromatin organization and gene expression
In mammals, heterochromatin is highly organized and closely associated with the lamina at the
nuclear periphery (Marshall and Sedat, 1999). Instead, actively transcribed regions (euchromatin)
appear to have a more random distribution in the nucleoplasm (Francastel et al., 2000). Recent
models of nuclear architecture describe lamins as determining factor for chromosome positioning
throughout the nucleus (Dorner et al., 2007; Vlcek and Foisner, 2007; Reddy et al., 2008). As a
consequence the lamins would be involved in anchoring chromatin to the nuclear lamina and would
also act as a nucleoplasmic scaffold for organizing chromatin in the nucleus.
The interaction between the lamins and chromatin involve their non-α-helical C-terminal tail
domain and the N- and C-terminal tail domains of core histones (Mattout et al., 2007; Goldberg et
17
al., 1999). Several proteins have been also described as lamin-binding proteins which directly link
both A- and B-types lamins to DNA (Schirmer and Foisner, 2007). Among these proteins are a
Lamin B Receptor (LBR), which is an integral protein of the INM (Ye and Worman, 1994), the
LAP2α protein, that binds specifically to Lamin A/C, the LAP2β protein, that interacts exclusively
with B-type lamins (Dechat et al., 2000; Furukawa and Kondo, 1998), and the
Autointegration Factor
Barrier to
(BAF), which has been reported to bind to dsDNA and to histones
(Margalit et al., 2007). However, the functional significance of these complex interactions remains
to be determined.
On a more global level, lamins could influence gene expression also because they may provide
a structural scaffold for the organization of RNA polymerase II transcriptional complexes but not
for RNA polymerase I- and III-directed transcription (Spann et al., 2002).
A-types lamins have been also described to interact with transcription factors thus regulating
gene expression. These interactions appear to regulate transcription in several ways: by sequestering
transcription factors in inactive complexes at the nuclear envelope, altering posttranslational
modifications important for their function and regulating transcriptional complexes (Andres and
Gonzalez, 2009).
1.2.5 Role of lamins in DNA replication and cell proliferation
Several lines of evidence indicate that lamins play a role in DNA replication. Indeed, a properly
assembled nuclear lamina is necessary to DNA replication activity (Ellis et al., 1997). It has been
shown that Lamin B1 colocalizes with the replication foci during late S phase in mouse 3T3 cells
(Moir et al., 1994) and that Lamin A/C are present at sites of early replication in normal human
fibroblasts (Kennedy et al., 2000). Functional lamins are needed for a correct localization of
replication factors such as PCNA and RFC, which are required in the elongation phase of
replication (Moir et al., 2000). The dependence of DNA replication on the nuclear lamina may be,
in part, mediated by the tumor suppressor Retinoblastoma product (pRb) since a complex formed by
pRb and Lamin A/C has been localized to replication foci (Ozaki et al., 1994). In consideration of
the physical interaction between Lamin A/C and pRb, there are clear evidences that Lamin A/C is
involved in the regulation of the cell cycle. pRb is a major cell cycle regulator that in its
hypophosphorylated state binds and inhibits the E2F transcription factors family necessary for cell
cycle progression (Giacinti and Giordano, 2006). Upon hyperphosphorylation of pRb, E2F is
18
released to initiate S phase. A role for lamins in this process is further suggested by the finding that
hypophosphorylated pRb is tightly associated with Lamin A/C-enriched nucleoskeletal preparations
of early G1 cells (Mancini et al., 1994). In addition, there is a dramatic reduction in pRb levels in
LMNA−/− fibroblasts, indicating that lamins are involved in regulating the turnover and
proteasomal degradation of pRb. Based on these findings, it has been suggested that the increased
rate of cell proliferation and the decreased capacity to undergo cell cycle arrest reported for Lamin
A/C-deficient cells is attributable to the destabilization of pRb (Johnson et al., 2004; Van Berlo et
al., 2005).
1.2.6 Laminopathies
Many aspects of nuclear as well as cytoplasmic activity are affected by modifications of the NE
and the nuclear lamina. Processes as fundamental as DNA replication, transcription, and cell
survival are altered in response to disruption of nuclear lamina, overexpression of mutant or
truncated lamins, and loss-of-function mutations of the LMNA gene. Given the diversity of
functions affected by these alterations, it is not surprising that complex patterns of tissue-specific
pathologies are associated with lamins defects in humans. At present, no mutations in the Lamin B
genes have been linked with human diseases, thus presuming that mutations in B-type lamins could
be embryonic lethal. Conversely, mutations in the LMNA gene has been directely associated with a
variety of human disorders broadly named “laminopathies” that are classified into primary and
secondary laminopathies. The primary laminopathies are caused by mutations in the LMNA gene.
The secondary ones are caused by mutations in the gene ZMPSTE-24 encoding for the endoprotease
FACE-1, enzyme which is required for the post-translational modification of the A-type lamins
(Ben et al., 2005).
The primary laminopathies are generally classified into five groups.
Group 1. This is the most frequent group and concerns diseases of skeletal and cardiac muscle.
The first mutations identified in the LMNA gene were those causing Autosomal-Dominant EmeryDreifuss Muscular Dystrophy (AD-EDMD) (Bonne et al., 1999). LMNA mutations display a wide
variability in the severity of the defects produced which could be lethal (Morris, 2001). However,
mutations in LMNA could also be responsible for dilated cardiomyopathy with conduction system
disease (DCM-CD1) without apparent dystrophy in the skeletal muscles (Fatkin et al., 1999). Limb
girdle muscular dystrophy 1B (LGMD1B) is also caused by mutations in LMNA, and is associated
19
with fewer cardiac complications and tendon contractures (Muchir et al., 2000). The heterogeneity
in phenotypes, even among members of a single family carrying the same LMNA mutation,
indicates that these disorders represent a spectrum of diseases that may originate from a common
genetic alteration and can be successively influenced by environmental factors. To date, diseases
affecting striated muscle comprise about 60% of the laminopathies. Considerably, about 50% of
patients diagnosed with AD-EDMD or EDMD have mutations in either the emerin (EMD) or LMNA
genes (Ben et al., 2005).
Group 2. The second group is associated with diseases in white adipose tissue and/or the
skeleton, such as Dunnigan-type familial partial lipodystrophy (FPLD) and Mandibuloacral
dysplasia (MAD) (Cao and Hegele, 2000) (Novelli et al., 2002). FPLD is inherited as an autosomaldominant trait mainly associated with a missense mutation of Arg482. FPLD is characterized by an
aberrant adipose tissue redistribution that may be a result of an autonomous defect in specific
subsets of mesenchymal or adipocyte precursors (Shackleton et al., 2000). Rare autosomal-recessive
mutations in the C-terminal domain of A-type lamins are responsible for MAD (Simha et al., 2003).
MAD is a disease with many of the metabolic and fat depot redistribution phenotypes of
lipodystrophy, but with a wide range of defects including alterations in skeletal development.
Group 3. The third group is a single autosomal-recessive mutation in the rod domain of A-type
lamins resulting in a peripheral neuropathy associated with demyelination of motor neurons, the
Charcot-Marie-Tooth syndrome type 2b (CMT2B1) (Chaouch et al., 2003). Families homozygous
for the R298C lamin variant have abolished deep-tendon reflexes, distal amyotrophy, motor deficits,
and loss of large myelinated nerve fibers. The muscular weakening associated with this disease is
likely a secondary result of loss of enervation and subsequent muscle atrophy. Interestingly,
neurons in the sciatic nerve of the LMNA null mice showed extensive demyelination (De SandreGiovannoli et al., 2002).
Group 4. The fourth group includes the premature aging diseases, Hutchinson Gilford Progeria
Syndrome (HGPS) and some cases of atypical Werner syndrome. HGPS is a rare dominantly
inherited disease associated with a splicing defect in exon 11 of the LMNA gene causing a 50 amino
acid truncation in the Lamin A protein without affecting the Lamin C protein (De SandreGiovannoli et al., 2003). The resultant Lamin A mutant has been named Progerin. Patients show
symptoms of premature aging, including severe growth retardation, loss of subcutaneous fat,
alopecia, reduced bone density, and poor muscle development. The average age of death in HGPS is
12 to 15 years, usually due to severe artherosclerosis resulting in myocardial infarction or stroke
(Sarkar and Shinton, 2001). A second premature aging syndrome is the autosomal-recessive
20
inherited Werner syndrome. Most (83%) patients have mutations at the WRN gene codifying for a
DNA helicase-exonuclease. A few (15%) but significant number of cases of atypical Werner
syndrome shows mutations in LMNA. These atypical patients have short stature, alopecia,
osteoporosis, lipodystrophy, diabetes, and muscle atrophy (Chen et al., 2003).
Group 5. The fifth group is a heterogeneous group of syndromes comprising overlapping
pathologies in different tissues described in the first four groups, suggesting a possible continuum in
the mechanisms underlying the laminopathies.
To date, over 200 mutations from more than 1000 individuals have been identified in the
LMNA gene, and a database on the nuclear envelopathies can be found at http://www.umd.be.
Fig. 1.9 Different mutations in LMNA are associated with different diseases. Most of the mutations resulting in the
diseases affecting striated muscles are distributed throughout the gene. The majority of the mutations that result in
Mandibuloacral disease, Dunnigan-type familial partial dystrophy, and Hutchinson Gilford Progeria syndrome cluster in
the region of the LMNA gene encoding the C-terminal globular domain.
21
1.2.7 Lamins and cancer
The expression of the A-type lamins is often reduced or absent in subsets of cells with a low
degree of differentiation and/or cells that are highly proliferating including human malignancies,
particularly leukemias, lymphomas, some skin cancers, including basal cell and squamous cell
carcinoma, adenocarcinoma of the stomach and colon, squamous and adenocarcinoma of the
esophagus, small cell lung cancer, testicular germ cell tumors and cancerous prostate tissues.
(Stadelmann et al., 1990) (Oguchi et al., 2002) (Venables et al., 2001) (Moss et al., 1999) (Broers et
al., 1993) (Machiels et al., 1997) (Coradeghini et al., 2006).
Altered expression and aberrant localization of A-type lamins often correlate with cancer
subtypes and cancer aggressiveness, proliferative capacity and differentiation state. Given that
tumor progression is often associated with regression from a more differentiated to a less
differentiated state, loss of Lamin A/C expression may not be surprising. Even though the altered
lamins expression are currently emerging as an additional event involved in malignant
transformation and tumor progression, the role of A-type lamins in the tumorigenesis is not yet
characterized and the molecular defect underlying the loss of A-type lamins in human cancer
remained unknown.
It has been reported that A-type lamins CpG island promoter hypermethylation is a significant
predictor of poor outcome in some lymphomas (Agrelo et al., 2005). Cancer may also be considered
an epigenetic disease and patterns of aberrant DNA methylation are now recognized to be a
common hallmark of human tumors. In fact, transcriptional inactivation by CpG island promoter
hypermethylation is a well-established mechanism for gene silencing in human tumors. Since Atype lamins present an important dual role as protector of chromatin from damage and as
multifunctional regulators of gene transcription, the epigenetic silencing of LMNA gene in
hematologic malignancies could aid understanding as to how lamins dysregulation could contribute
to cellular transformation.
22
2
AIMS
Considering the involvement of Lamin A/C in the tissue differentiation during the embryonal
development, the main objective of this work was to investigate whether Lamin A/C could regulate
neuroblastoma differentiation. Neuroblastoma is a highly aggresive embryonic tumor of the
autonomic nervous system and is the most common extracranial solid cancer in childhood.
Moreover, taking into account the significance of differentiation stage in the neuroblastoma
tumor progression I intended to identify a role of Lamin A/C in the tumorigenesis of this neuronal
cancer and to investigate whether A-type lamins could also represent a marker of drug response.
To this aims I chose a neuroblastoma model able to differentiate in vitro and showing high
expression levels of Lamin A/C. As differentiating stimulus I used the all-trans retinoic acid, the
most effective compound which has been shown to induce differentiation in neuroblastoma cells
and that is employed as biological agent in clinics to improve patients‟ survival.
23
3
MATERIALS AND METHODS
3.1 Cell lines maintenance, differentiation and treatments
Human malignant neuroblastoma (NB) SHSY5Y cell line was purchased from the American
Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in a 1:1 mixture of
Eagle's Minimum Essential Medium and F12 Medium (Gibco, Grand Island, NY, USA)
supplemented with 10% fetal bovine serum (FBS, Hyclone), 2 mM L-glutamine, 0.5% nonessential amino acids, 0.5% sodium pyruvate and 1% penicillin and streptomycin in a fullyhumidified incubator containing 5% CO2 at 37˚C. LAN-5 NB cell line was a gift of Dr. Doriana
Fruci and was grown in RPMI-1640 (Gibco, Grand Island, NY, USA) supplemented with 10% FBS,
2 mM L-glutamine, 100 U/L penicillin and 100μg/L streptomycin in a fully-humidified incubator
containing 5% CO2 at 37˚C.
For differentiation experiments cells were seeded at a density of 5×103 cells/cm2. The
following day cells were induced to differentiate by 10 µM retinoic acid (ATRA; Sigma Chemical,
St. Louis, MO, USA, named RA throughout the thesis) in a “differentiation medium” composed by
50% fresh and 50% conditioned culture medium. Cells were fed after 3 days with differentiation
medium containing fresh RA. RA was dissolved in dimethyl sulfoxide (DMSO, Sigma Chemical, St.
Louis, MO, USA) and stored as stock at -80˚C. Because of light sensitivity of RA, all incubations
were performed under subdued lighting. The concentration of DMSO in each experiment was
always ≤0.01%, which was not toxic and did not induce differentiation.
For drugs experiments cells were seeded at a density of 5×103 cells/cm2. Twenty-four hours
after plating, fresh medium was added and after further 24 hours (48 hours of growth) cells were
treated with different doses of cis-platin (DDP) or etoposide (VP-16), both dissolved in DMSO , for
2 hours.
3.2 Generation of recombinant viruses and lentiviral infection of cells
LMNA-Knock Down SHSY5Y cell line (named miRL) was generated by using a lentiviral
expression vector
containing a double strand oligo encoding a pre-miRNA sequence as
“knockdown cassette” thus enabling the expression of engineered miRNA sequence from Pol II
promoters directed versus the LMNA mRNA. The knockdown cassette contains specific miRNA
flanking sequences that allow proper processing of the miRNA. The expression vector design is
based on the miRNA vector system developed in the laboratory of David Turner (U.S. Patent
24
Publication No. 2004/0053876) and includes the use of endogenous murine miR-155 flanking
sequences. The lentiviral expression vector was produced using the BLOCK-iT Lentiviral Pol II
miR RNAi Expression System (Invitrogen,, San Giuliano Milanese, Milan, Italy) to obtain from the
pcDNA6.2-GW/EmGFP-miR-LMNA (Invitrogen) a Gateway®-adapted lentiviral destination vector
(pLenti6/V5-DEST vector) according to the manufacturer‟s instructions. As a negative control we
used the pcDNA™6.2-GW/± EmGFP-miR-neg control plasmid that contains an insert that can form
a hairpin structure that is processed into mature miRNA, but is predicted not to target any known
vertebrate gene.
Briefly, to transfer the pre-miRNA cassette (named, LMNA pre-miRNA cassette or Neg CTR
pre-miRNA cassette) from pcDNA™6.2- GW/+EmGFP-miR expression clone into pLenti6/V5DEST vector, we performed two Gateway® recombination reactions as follows. An “entry clone”
was generated by performing a BP recombination reaction between the attB substrate
(pcDNA™6.2-GW/EmGFP-miR expression clone) and attP substrate (pDONR™221 vector) using
BP Clonase™ II Enzyme Mix. Next, an LR recombination reaction between the resulting “entry
clone” (attL substrate) and pLenti6/V5-DEST vector (attR substrate) was performed using LR
Clonase™ II Enzyme Mix, obtaining the pLenti6/V5-GW/LMNA miRNA and the pLenti6/V5GW/Neg CTR miRNA expression plasmids.
In order to produce lentiviral stocks, these vectors (3 µg) and the ViraPower™ Packaging
Mix (9 µg) were cotransfected into 6x106 packaging 293FT cells using Lipofectamine 2000
(Invitrogen Corp), according to the manufacturer‟s instructions. The supernatants were collected
after 48 and 72 hours, and used for infection.
SHSY5Y cells were seeded into 6-well plates at a concentration of 2×105 cells/well and the
following day were infected for 18 hours with virus supernatant from pLenti6/V5-GW/LMNA
miRNA or pLenti6/V5-GW/Neg CTR miRNA and then fresh medium was added. After 6 hours
cells were subjected to a second round of infection for additional 18 hours. Stably transduced cells
were selected by culturing in presence of blasticidin 3 µg/mL (Invitrogen). The transduced cells
were screened by western blotting and real-time PCR assays to determine the levels of LMNA
expression.
25
3.3 Western Blot analysis
Cultured cells were washed 2 times with PBS 1X and then incubated 1 min in urea buffer (8
M urea, 100 mM NaH2PO4, 10 mM Tris pH 8), scraped, harvested and then briefly sonicated.
Proteins were subjected to SDS–polyacrylamide gels electrophoresis. The resolved proteins were
blotted overnight to nitrocellulose membranes, which then were blocked in PBS 1X containing 5%
non-fat milk for at least 1 hour. Blots were incubated with the following primary anti-human
antibodies: anti-lamin A/C polyclonal antibody (N-18; Santa Cruz Biotechnology); anti-tyrosine
hydroxyase polyclonal antibody (Cell Signaling); anti-GAPDH monoclonal antibody (6C5,
Chemicon). The membranes were then incubated 45 min with the relevant secondary antibody (anti
mouse, anti-rabbit or anti-goat IgG) conjugated with Alexa fluor 680 (Invitrogen) or IRDye 800
(LI-COR Biosciences) and analyzed with the Licor Odyssey Infrared Image System in the 700 or
800 nm channel.
3.4 Immunofluorescence microscopy
Cells were seeded on coverglass supports in complete medium and treated with ATRA for the
indicated times. Cells fixed with 4% (w/v) paraformaldehyde were permeabilized in PBS containing
0.1% Triton-X100. NF200 was detected using the anti-NF200 monoclonal antibody (N52; Sigma).
Alexa Fluor 594 goat anti-mouse was used as secondary antibody. Antibodies were diluted in PBS.
The nuclei were stained with 1 mg/ml DAPI for 5 min in PBS. Finally, cells were washed in PBS,
briefly rinsed in ddH2O and glasses were mounted in ProLong Gold anti-Fade Reagent (Molecular
Probes). Images were acquired through a confocal laser scanning microscope (Leica Confocal
Microsystem TCS 566 SP5). Images were processed using Leica Application Suite 6000.
Brightness and contrast were adjusted. Figures were generated using Adobe Photoshop 7.0 and
Adobe Illustrator 10.
3.5 Total RNA preparation
Cells were seeded in complete medium and after the different treatments were harvested and
total RNA was isolated using NucleoSpin RNA II silica columns (Macherey-Nagel). RNA quantity
was determined by absorbance at 260 nm using a NanoDrop UV-VIS spectrophotometer. Quality
and integrity of each sample was checked using the Agilent BioAnalyzer 2100 (Agilent RNA 6000
26
Nanokit): samples with a RNA Integrity Number (RIN) index lower than 8.0 were discarded
(Schroeder et al., 2006).
3.6 Real‐time RT‐PCR analysis
RNA (500 ng) was retro-transcribed with High-Capacity cDNA Reverse Transcription Kit
(Applied Biosystem) according to the manufacturer‟s instructions. Equal amount of cDNA was then
subjected to real time PCR analysis with an Applied Biosystems 7900HT thermal cycler, using the
SensiMix SYBR Kit (Bioline) and the following specific primers at a concentration of 200 nM:
LMNA
(unigene
Hs.594444)
F:
AGCAAAGTGCGTGAGGAGTT
and
R:
AGGTCACCCTCCTTCTTGGT; TH (unigene Hs.435609) F: ACGCCAAGGACAAGCTCA and
R:
AGCGTGTACGGGTCGAACT;
GAPDH
(unigene
Hs.544577)
F:
AGCCACATCGCTCAGACA and R: GCCCAATACGACCAAATCC; TBP (unigene Hs.590872)
F: GAACATCATGGATCAGAACAACA and R:
(unigene
Hs.356331)
F:
ATAGGGATTCCGGGAGTCAT; PPIA
ATGCTGGACCCAACACAAAT
and
R:
TCTTTCACTTTGCCAAACACC. Each experiment was done in technical quadruplicates.
Expression data were normalized using the Ct values of the internal controls GAPDH, TBP and
PPI1A.
3.7 Whole genome expression profiling
The gene expression profiling was performed using the Agilent one-color microarray
System (http://www.chem.agilent.com/enUS/Products/Instruments/dnamicroarrays/Pages/default.a
spx). Poly A+RNA (500 ng) was retrotranscribed using oligo-dT primers linked to the T7- promoter
and the resulting cDNA was used as a template for cyanine 3- CTP labelled cRNA preparation,
using the Agilent Low Input Linear Amplification Kit. The labeled cRNA was purified with
Qiagen‟s RNeasy mini spin columns. To monitor both the labelling reactions and the microarray
performance, Agilent Spike-In Mix was added to the mRNA samples prior to labelling reactions
according the RNA Spike-In protocol. Cyanine 3 labelled cRNA were hybridized to Agilent 4x44K
whole human genome oligonucleotide microarray (GE2-v4_91). In this library the number of
mRNAs is larger than the total number of genes, since in many cases different mRNAs probes (1 to
5) target the same gene. Agilent microarray probes are 60-mer DNA oligonucleotides and contains
41000 unique biological genes and transcripts. Microarray hybridizations were carried out in
27
Agilent‟s SureHyb Hybridization Chambers containing 1650 ng of Cyanine 3-labelled cRNA per
hybridization. The hybridization reactions were performed at 65°C for 17 hours using Agilent‟s
Gene Expression Hybridization Kit. The hybridized microarrays were disassembled at room
temperature in Agilent Gene Expression Wash Buffer 1. After the disassembly, the microarrays
were washed in Gene Expression Buffer 1 for one minute at room temperature, followed by
washing with Gene Expression Wash Buffer 2 for one minute at 37°C. The microarrays were then
treated with acetonitrile for one minute at room temperature. Post-hybridization image acquisition
was accomplished using the Agilent Scanner G2564B, equipped with two lasers (532 nm and 635
nm) and a 48 slide auto-sampler carousel. Data extraction from the 20 bits TIFF images was
accomplished by Agilent Feature Extraction ver 10.1 software using the standard Agilent one-color
gene expression extraction protocol (GE1_107_Sep09). For all the subsequent analysis, the
“gProcessedSignal” data column of the output file was used, containing the median signals
corrected by spatial and multiplicative detrend.
Data filtering was performed in Microsoft Excel using any of the following criteria to
discard spots: spots with more than 5% of saturated pixel in any of the two channels, spots with a
Signal/Noise ratio smaller than 3 in any of the two channels, where Signal = (median of the spot median spot background level) and Noise is the Standard Deviation of the median spot background.
Differential gene lists were obtained from filtered data using Agilent GeneSpring GX 7.3 and
Microsoft Excel. A threshold of 100.0 in the absolute expression level was chosen to obtain a higher
stringency. Differentially expressed genes were identified as those showing an average fold change
ratio larger than 1.5 and lower than 1/1.5 in the linear scale.
Functional annotations of differential gene lists was performed using the DAVID web tool
(http://david.abcc.ncifcrf.gov/) (Huang et al., 2009), selecting the following categories both for
functional clustering (high stringency) and charts: goterm_bp_4, goterm_bp_5, goterm_cc_4,
goterm_cc_5,
goterm_mf_4,
goterm_mf_5,
panther_bp_all,
panther_mf_all,
interpro,
pir_superfamily, smart, panther_family, panther_subfamily, kegg_pathway, panther_pathway,
chromosome, cytoband, cog_ontology, sp_pir_keywords, up_seq_feature. Gene clustering analysis
was done using MeV 4.4 (MultiExperiment Viewer, TM4 suite , http://www.tm4.org/mev/) (Saeed
et al., 2006).
Subsets of differential genes associated to specific cancer features were selected, based on
the gene annotations in the Cancer Gene Index of the National Cancer Institute (NCI).
database
is
a
file
in
XML
format
that
can
be
downloaded
from
the
The
URL:
https://cabig.nci.nih.gov/inventory/data-resources/cancer-gene-index/. The current Index version
28
(June 2009)
is a collection of records on about 7000 human genes identified as having an
association with cancer, based on data mining of Medline. A gene is included in the database if the
name co-occurs in a single Medline sentence with a cancer disease or compound/treatment term
found in the NCI Thesaurus. The gene symbols are those defined by the HUGO Gene Nomenclature
Committee, and the annotations of Agilent microarray probes are updated to 15 January 2010. We
have compiled three gene symbol lists by extracting, from the whole Cancer Gene Index, genes
associated to the following three Boolean queries, inserted as Regular Expressions in the search tool
of a pure text editor (TextPad): List1: “migration OR invasion OR invasive OR metastasis OR
adhesion OR adhesive”; List2: “drug resistance OR drug resistant OR survival OR survive OR
sensitive OR sensitivity”; List3: “aggressive OR aggression OR progression OR progressive”. A
gene was included in out lists if any of the terms in the corresponding query appeared in the gene
annotation records. In particular most of the search terms in these lists appeared within the
annotations tags <Statement>, <Comments> or <MatchedDiseaseTerm>. The overlap between the
three gene lists is shown in the Venn diagram below.
List1
List2
228
584
514
986
295
210
437
List3
Fig. 3.1 Venn diagram of the three gene lists obtained from the Cancer Gene Database of the National Cancer Institute
(NCI). Numbers correspond to gene symbols in the different set regions.
We selected for specific analysis subsets of genes differential in our experiments and with a match
in at least one of the three lists List1, List2 or List3.
29
3.8 Plating Efficiency Assay
One thousand cells were seeded at clonal density on a 35 mm Ø dish in complete medium.
After 10 days from seeding, cells were fixed for 1 hour with a solution of 2% methilene blue in 96%
ethanol. Dishes were then accurately washed with double distilled H2O and colonies were counted.
The number of colonies formed was expressed as percent of colonies with respect of cells seeded. A
total of three independent experiments were performed.
3.9 In vitro Cell Migration Assay
The assay was performed in Boyden chambers. Cells (8 x 105 cells/800 µl) were added to
the upper chamber in complete medium without FBS. The lower compartment was filled with 200
µl of complete medium containing 10% FBS, or with medium supplemented with 0.1% bovine
serum albumin (BSA) to evaluate random migration (negative control). The compartments were
separated by an 8-mm pore size polycarbonate filter (Costar Corp., Cambridge, MA), coated with
gelatin (5 µg/ml; Sigma, Milan, Italy) for chemotaxis. After incubation for 6 hours in a humidified
5% CO2 atmosphere at 37°C, cells on the upper side of the filter were removed mechanically and
cells that had migrated to the lower surface of the filter were fixed in ethanol and stained with
crystal violet solution (2% crystal violet, 10% ethanol) for 30 min. Cells were washed twice with
distilled water, 200 µL of a 10% acetic acid solution was added, and cells were incubated at room
temperature for 1 hour. Absorbance was measured at 570 nm. Each migration was tested in
quintuplicate and experiments were repeated at least three times.
3.10 Zymography of Gelatinolytic Activity
Subconfluent cells were incubated for 24 hours in complete medium containing 1% FBS.
Supernatants were collected and centrifuged (2000 g, 10 min, 4°C) to remove cellular debris. The
conditioned media (CM) of cells were concentrated with Centricon-30 concentrators (Amicon,
Danvers, MA). Each sample derived from 4x103 cells was applied to SDS-PAGE on a 10%
polyacrylamide gel with 0.2% (w/v) gelatin. Gel electrophoresis was performed under non-reducing
conditions without boiling. The gel was rinsed twice for 30 minutes in 2.5% (v/v) Triton X-100 to
remove SDS and renature the proteins and incubated with activation buffer (50 mM Tris-HCl pH
7.6; 5 mM CaCl2;1 µM ZnCl2; 1% Triton X-100) overnight at 37°C with constant shaking. The gel
30
was stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 in 50% (v/v) methanol and 10% (v/v)
acetate and then destained in 10% (v/v) methanol and 10% (v/v) acetate. Enzymatic activity was
detected as a white band on the resulting blue background of undigested gelatin.
3.11 P-Glycoprotein immunostaining
Cells (106/sample) were washed in Washing Buffer (WB, PBS; 10 mM NaN3; EDTA
0.002%) and purified anti-P-Glycoprotein mouse (4E3.16, Calbiochem) or control IgG, diluted in
complete medium at 12.5 μg/mL, were added to samples and incubated for 1 hour on ice. Cells
were washed twice with WB and incubated in WB containing goat anti-mouse RPE (Southern
Biotec Associates) for 40 min. After washing twice in WB, cells were resuspended in 300 μl of PBS
for flow cytometric analysis. Ten thousand events per sample were acquired by using a FACScan
cytofluorimeter.
3.12 Calcein/AM Retention Assay
Cells (5x105/sample) were suspended in 1 mL of complete medium and incubated with or
without Verapamil (5 µM, Sigma–Aldrich USA) for 30 min at 37 oC. Then cells were washed two
times in PBS containing 0.2% BSA to eliminate Verapamil retained in cells. Washed cells were
suspended into the same medium and incubated with Calcein/AM (100 nM, Sigma–Aldrich USA)
for 20 min at 37 oC. Finally, cells were washed twice in PBS containing 0.2% BSA, cooled to 4 oC
and analysed by FACS. Twenty thousand events per sample were acquired by using a FACScan
cytofluorimeter.
31
4
RESULTS
4.1 Silencing of LMNA gene inhibits RA-induced differentiation in SHSY5Y
cell line
To investigate whether Lamin A/C could play a role in neuroblastoma differentiation, the
SHSY5Y and LAN-5 neuroblastoma cell lines, showing different expression levels of Lamin A/C,
were induced to differentiate for 3, 5 and 7 days in response to 10 µM RA. SHSY5Y cells increased
the expression of Lamin A/C upon induction with RA as evaluated at protein and mRNA level.
Conversely, LAN-5 cells showed barely detectable expression levels of Lamin A/C protein and of
LMNA mRNA, with no variation during the induced differentiation (Figure 4.1 A and B).
Fig. 4.1 Increased Lamin A/C expression during RA-induced differentiation in SHSY5Y cell line. A) Levels of
Lamin A/C protein in the SHSY5Y and LAN-5 neuroblastoma cell lines untreated or after 10 µM RA treatment for 3, 5
and 7 days (d) as detected by Western blot analysis using anti-Lamin A/C N18 polyclonal antibody. Each lane was
loaded with 30 µg of proteins from cell lysate. GAPDH was used as a loading control. The experiment was repeated
three times showing similar results. Representative blots are presented. B) Quantitative RT-PCR analysis of LMNA
mRNA levels in SHSY5Y (white) and LAN-5 (black) cells untreated or after 10 µM RA treatment for 3, 5 and 7 days.
The amounts of LMNA mRNA were normalized to GAPDH housekeeping gene. Data are reported as mRNA
quantification relative to SHSY5Y untreated cells and are average of three separate experiments. Bars represent
standard deviation. NT, untreated cells.
32
To gain insight into the function of Lamin A/C in neuroblastoma differentiation, we silenced
LMNA gene in SHSY5Y cell line and studied the effects during the differentiation processes. LMNA
knock-down was obtained by infecting the cells with a lentiviral vector carrying an artificial
miRNA targeting the LMNA mRNA (see Materials and Methods). The LMNA silenced SHSY5Y
cells, named miRL cells throughout the thesis, show a reduction in Lamin A/C expression of about
70% compared to control (CTR) cells infected with a control vector (Figure 4.2 A). The reduced
Lamin A/C expression resulted in the impairment of neuronal differentiation as indicated by the
inhibition of the formation of neuritic processes in miRL compared to CTR cells differentiated for 5
days (Figure 4.2 B).
Fig. 4.2 Silencing of LMNA in SHSY5Y cell line during RA-induced differentiation. A) Levels of Lamin A/C
protein in SHSY5Y CTR and miRL cells untreated or after 10 µM RA treatment for 3, 5 and 7 days as detected by
Western blot analysis using anti-Lamin A/C N18 polyclonal antibody. Each lane was loaded with 30 µg of proteins
from cell lysate. GAPDH was used as a loading control. The experiment was repeated three times showing similar
results. A representative blot is presented. B) Inverted light microscope images of CTR and miRL cells untreated or
after 10 µM RA treatment for 5 days.
33
Consistent with the outgrowth inhibition of neurites is the decreased expression in miRL cells
of the neuronal differentiation marker Neurofilament-200 (NF200), the intermediate filament that
provides structural support for the neuritic processes (Figure 4.3 A). The impairment of
differentiation in miRL cells is further confirmed by the loss of induction of the noradrenergic
differentiation marker tyrosine hydroxylase (TH), as evaluated by Western blot analysis and qRTPCR (Figure 4.3 B and C).
Fig. 4.3 Silencing of LMNA impairs the RA-induced differentiation in SHSY5Y cell line. A) Representative
confocal images of CTR and miRL cells untreated (NT) or after 10 µM RA treatment for 5 days, immunostained with
anti-NF200 N52 monoclonal antibody (red) and with the DNA dye DAPI (blue). B) Levels of tyrosine hydroxylase (TH)
protein in CTR and miRL cells NT or after 10 µM RA treatment for 3 and 5 days as detected by using anti-TH
polyclonal antibody. Each lane was loaded with 30 µg of proteins from cell lysate. GAPDH was used as a loading
control. The experiment was repeated three times showing similar results. A representative blot is presented. B)
Quantitative RT-PCR analysis of TH mRNA levels in CTR (white) and miRL (black) cells untreated or after 10 µM RA
treatment for 1 and 2 days. The amounts of TH mRNA were normalized to GAPDH housekeeping gene. Data are
reported as mRNA quantification relative to CTR NT cells and are average of three separate experiments. Bars
represent standard deviation.
34
4.2 Gene expression profiling of SHSY5Y CTR and miRL cells reveals an
impairment of differentiation
To get insight into the impairment of cell differentiation produced by the LMNA silencing in
SHSY5Y cells, we compared the gene expression profile of CTR and miRL cells both in RA treated
and untreated cells, using the Agilent microarray technology. As concerns the gene expression
profiling in the two cell lines during the differentiation, it was determined at day 5 of RA treatment.
A hierarchical cluster of expression data was obtained from the one-color microarray experiment
(Figure 4.4). The set used for clustering is composed by the 1801 genes that are modulated at least
1.5 fold by retinoic acid in both CTR and miRL cells (see intersection set in the next Figure 4.5 A).
The 4 columns correspond to the four samples. As expected, the sample tree on the top shows that
the untreated and the RA treated samples behave differently, since the two untreated samples and
the two RA treated ones cluster together in couple.
To investigate the differential gene expression between CTR and miRL RA-treated cells we
defined two sets of genes obtained comparing RA-treated samples with untreated ones in CTR
(CTR RA/CTR NT), or in miRL (miRL RA/miRL NT ) cells. The Venn diagram in Figure 4.5
shows that 1782 genes are modulated by RA only in CTR cells, with 247 up-regulated genes and
1535 down-regulated genes, while 1131 are the genes modulated only in miRL cells, with 694 upregulated and 437 down-regulated genes. A number of 1801 genes are regulated during RA
induction independently of LMNA expression both in CTR and in miRL cells.
To gain more insights into the possible mechanisms underlying differential gene expression
patterns, functional analysis of differential gene lists was performed by the DAVID web tool, to
find statistically over-represented functional groups. The main statistically significant (p < 0.05)
functional categories modulated during differentiation in CTR cells are listed in Table 1, among
them “nervous system development” for up-regulated genes and , “DNA replication”, “nuclear
division” and “regulation of cell cycle” for down-regulated genes. Similarly, the main statistically
significant (p < 0.05) functional categories modulated by RA only in miRL cells are listed in Table
2: the list includes “metallothionein”, “cell migration” and “angiogenesis” for up-regulated genes
and “nervous system development”, “neurogenesis” and “generation of neurons” for downregulated genes.
35
Fig. 4.4 Hierarchical cluster of expression data obtained from the one-color microarray experiment. The set used
for clustering is composed by the 1801 genes that show an average ratio in the linear scale either greater than 1.5 or
smaller than 1/1.5, both for (miRL RA/miRL NT) and (CTR RA/CTR NT) ratios (see intersection set in light grey in
Fig. 4.5A). Each gene is visualized as a single row and each sample is represented by a single column. Euclidean
distance, with average linkage, is the metric used for clustering. The rainbow color scale indicate the Log2 absolute
expression of the genes, with lighter colors noting higher levels of expression, where the color scale ranges from 15.0 to
7.0 as shown in the scale bar on the right of the figure. Only microarray probes with official gene symbol annotation
were used. CTR, SHSY5Y control cells; miRL, LMNA silenced SHSY5Y cells; RA 5d, RA treated cells for 5 days.
36
Fig. 4.5 Venn diagram and histograms of the sets of differentially expressed genes between CTR e miRL cells
after RA treatment. Venn diagram of two sets of differentially expressed genes, showing an average relative ratio
greater than 1.5 or smaller than 1/1.5 in the linear scale: the CTR* set, obtained comparing RA-treated samples with
untreated (NT) ones in CTR cells, (CTR RA /CTR NT) ratio, is composed by a total of 3583 genes, while the miRL*
set, obtained comparing RA-treated samples with NT ones in miRL cells, (miRL RA/miRL NT) ratio, is composed by a
total of 2932 genes. The {CTR*-miRL*} set, in white, is composed by 1782 genes modulated by RA only in CTR cells,
with 247 up-regulated genes and 1535 down-regulated genes. The {miRL*-CTR*} set, in dark grey, is composed by
1131genes modulated by RA only in miRL cells, with 694 up-regulated and 437 down-regulated genes. In the two
histograms, up-regulated genes are in red and down-regulated genes are in green.
37
4.3 The impairment of cell differentiation in LMNA silenced SHSY5Y cells is
associated with increased tumor progression
Since undifferentiated neuroblastoma tumors are related to a worse clinical outcome we have
studied whether the impairment of cell differentiation occurred in LMNA silenced cells could be
associated to a more aggressive phenotype. We first compared the expression profiles of
undifferentiated CTR and miRL cells. The genes whose expression was down-regulated by LMNA
silencing were 636, while those whose expression was up-regulated were only 100 (Table 3). In
order to deeper investigate a possible link between Lamin A/C and tumor progression we defined a
list of genes associated to specific cancer features (e.g. metastasis, drug resistance, and tumor
progression) obtained using the Cancer Gene Index database of the NCI. Comparing the whole gene
expression profile of miRL cells versus CTR cells to the gene annotations in the Cancer Gene Index,
we found a differential expression in 43 genes (15 up-regulated and 28 down-regulated) which were
related to the more aggressive phenotype. Among these we found down-regulated tumor suppressor
genes (H19, TSPAN13, APC2, INHBA, SYK, TFAP2B, SPRY2, FHIT, NEURL, KISS1R, SP100,
PTCH1, CASP2) and up-regulated metalloproteases (MMP9, MMP15), angiogenic factors (ENPP2)
and metastases-associated genes (MALAT1, ETV5, CTHRC1) (Fig. 4.6 and Supplementary table
S.1).
We then investigated how such gene modifications could affect some biological features which
are characteristic of a more aggressive tumor phenotype. First, we studied the clonogenic ability of
the CTR and miRL SHSY5Y cell lines by evaluating the plating efficiency of the two cell lines. An
increase of more than 50% in the ability to form colonies was obtained in the miRL compared to the
CTR cells (Figure 4.7 A). As second point we analyzed the motility of the two cell lines, since an
increase of this cell function is strictly associated to the tumor progression. Silencing of LMNA gene
significantly increases the migration activity of SHSY5Y cells in response to FBS 10% as
compared to the CTR cells. Whereas, migration in the absence of chemoattractant (negative control)
is limited (Figure 4.7 B).
Tumor progression involves also the acquisition by tumor cells of the capability to degrade the
basement membrane thus invading the surrounding environment and, in general, remodeling the
extracellular matrix. Thus we examined whether LMNA silencing could lead to an increased
protease secretion by the tumor cells. In the zymographic analysis of the conditioned media from
SHSY5Y cells, lower levels of secreted 72-kDa gelatinase (also designed MMP2) were found in
CTR than in miRL cell cultures (Figure 4.7 C).
38
Fig. 4.6 Relative expression of tumor aggressiveness related genes in miRL versus CTR cells. Directional fold
change values for a subset of genes, related to tumor aggressiveness, identified in microarray experiment as either upregulated (red bars) or down-regulated (green bars), when comparing LMNA silenced SHSY5Y cells versus CTR cells.
Gene symbols are indicated on the left (specifications are reported in Supplementary Tables S.1). The fold-change (fc)
values are in the linear scale. To visualize changes relative to the down-regulated genes, they were converted in
negative values using the formula (-1/fc). This gene set was selected according to the annotations in the NCI Cancer
Gene Index database, combining specific gene lists obtained from the database (see Materials and Methods).
39
Fig. 4.7 Silencing of LMNA induces a more aggressive tumor phenotype in SHSY5Y line. A) Plating efficiency of
CTR and miRL cells referred as percent of colonies formed 10 days after seeding. B) Migration assay in CTR and miRL
cells. Single cell suspensions in DMEM supplemented with 0.1% BSA were incubated for 6 hours on top of gelatincoated filters. The migration was evaluated by filling the lower compartment with medium containing 10% Fetal
Bovine Serum (FBS). DMEM supplemented with 0.1% BSA was used as negative control to evaluate random migration.
C) Analysis of metalloproteases secretion in CTR and miRL cells. Conditioned media of both cell lines, incubated for
48 hours in medium containing 1% FBS, were collected and analyzed for gelatin zymography. Equivalent amounts of
proteins were loaded. White bands against a dark background correspond to the gelatinolytic areas of MMP2 (72 kDa)
activity. Below is reported the densitometric quantification of the bands.
40
4.4 Low levels of Lamin A/C expression are associated with increased
resistance to chemotherapeutic agents
Since tumor progression is generally associated with a reduced cellular ability to respond to
antineoplastic treatments, we also investigated whether A-type lamins could represent a marker of
sensitiveness to conventional antitumoral therapy. Therefore we studied the effect of Cisplatin
(DDP) and Etoposide (VP-16) drugs on the cell survival of CTR and miRL SHSY5Y cell lines.
Colony formation ability was assayed after treating the cells with different doses of the two drugs.
Silencing of LMNA resulted in a significant increase of cell survival values for both compounds
(Figure 4.8 A). This increased drug resistance in LMNA silenced cells is consistent with the
augmented expression of the efflux/conditional transporter P-glycoprotein (Pgp), that belongs to the
ABCB subgroup of the ATPBinding-Cassette (ABC) transporter superfamily and is known to
confer resistance to a broad variety of structurally unrelated chemotherapeutic agents and to restrict
bioavailability of many therapeutic drugs in experimental models (Figure 4.8 B). The increase in
Pgp expression at the cell surface resulted in higher Pgp functional activity as assessed by
alterations in calcein accumulation, a probe which is widely used to evaluate Pgp functionality
(Figure 4.8 C). Thus the larger amounts of Pgp detected by flow cytometry in miRL cells are likely
to represent quantity of Pgp available for drug efflux.
41
Fig. 4.8 Silencing of LMNA is associated with increased resistance to chemotherapeutic agents. A) Effect of cisplatin (DDP; left panel) or etoposide (VP-16; right panel) in CTR and miRL cells as evaluated by colony forming assay.
Cell survival data are reported as percent of control and are average of three different experiments with similar results.
Bars represent standard deviation. Black square, miRL; open diamond, CTR. B) Flow cytometry analysis of Pgp
expression as evaluated by immunofluorescence using a monoclonal antibody recognizing an external epitope of the
Pgp. In the left panel are reported the flow cytometry histograms of negative control (Neg, mouse IgG), CTR and miRL
cells after immunostaining. In the right panel are reported the Pgp positive cells in the CTR and miRL samples relative
to the negative control. C) Pgp functionality using calcein assay. Calcein mean fluorescence values in each sample were
referred to the relative control. Verapamil 10 µM was used as competitive inhibitor of the Pgp function to obtain a
directed increase of calcein uptake. Data are average of three different experiments with similar results. Bars represent
standard deviations.
42
5
DISCUSSION
We studied the effect of Lamin A/C down-regulation on the differentiation processes of
neuroblastoma cells. The degree of neuroblastoma differentiation has been related with a favorable
prognosis of the patients. However, the mechanisms governing such differentiation are only
partially understood. The ability of many neuroblastoma cell lines to activate neuronal
differentiation in vitro in the presence of various agents, such as retinoids, allowed studying key
differentiation properties of neuroblastoma cells which are in common with primary neurons.
Our working hypothesis came from an initial observation that various neuroblastoma cell lines
express different levels of Lamin A/C protein. Among the cell lines able to differentiate we then
chose the noradrenergic neuroblastoma cell line (SHSY5Y) with the highest expression of Lamin
A/C, and we silenced the LMNA gene codifying for Lamin A/C in order to study the effects on the
differentiation of such cells. Our results show that silencing of LMNA in SHSY5Y cells inhibited
their ability to differentiate and gave rise to a more aggressive tumor phenotype thus demonstrating
a central role of Lamin A/C in the neuroblastoma tumor differentiation and progression.
We show that Lamin A/C expression increases in SHSY5Y cells during the differentiation
program induced by retinoic acid. Our results are in agreement with different studies on the early
development of Xenopus as well as of Drosophila and chicken embryos showing that the nuclear
lamina composition correlate with major programs in cell differentiation (Stick and Hausen, 1985)
(Lehner et al., 1987) (Riemer et al., 1995). Also in mammals various authors showed that the
expression of A-type lamins is regulated according to developmental stage of tissue differentiation
(Benavente et al., 1985) (Guilly et al., 1990) (Rober et al., 1989) (Mattia et al., 1992) (Lin and
Worman, 1997). In addition, in a more recent study Constantinescu and colleagues showed that
Lamin A/C expression is an early indicator of embryonal stem cell differentiation (Constantinescu
et al., 2006). In particular, it has been recognized as marker of muscle and adipocyte differentiation
(Favreau et al., 2003) (Frock et al., 2006) (Lloyd et al., 2002).
For the first time we demonstrate that Lamin A/C is necessary for the differentiation of
neuroblastoma tumor cells. Indeed, silencing the LMNA gene in SHSY5Y cells produced the
inhibition of the differentiation program as firstly evidenced by the reduced cellular ability to form
mature neurites. In fact, neurites are normally outgrown from neurons functional networks
connecting neurons each other through synapses and allow the interneuronal transmission of
electrical or chemical signals thus rendering differentiated neurons one of the main functional cell
type of the nervous system. Further confirmation of the differentiation impairment is represented by
43
the decreased levels of the rate-limiting enzyme in noradrenalin synthesis, tyrosine hydroxylase
(TH). In fact, as also reported by Kume and colleagues undifferentiated noradrenergic SHSY5Y
cells cannot produce noradrenalin because they do not express TH (Kume et al., 2008). Consistent
with the inhibition of cell differentiation occurred in LMNA silenced cells are the data from the gene
expression profiling. Microarrays allowed us to measure the expression of thirty-one thousand
genes. Among them we first chose those genes that are co-modulated by a differentiation stimulus
in CTR and miRL cells and we correlated them basing on hierarchical clustering. This approach
allowed us to group together the untreated or retinoic acid treated cells evidencing a good
clusterization of microarray data with the cellular response to a differentiation stimulus. Even
though the LMNA silenced cells did not differentiate, it is likely that the signaling pathway engaged
by retinoic acid is generally still functioning in miRL cells. Their different response to the
differentiating agent could be ascribed to reduced activity of Lamin A/C in various pathways related
to transcription, preventing the expression of genes necessary for the completion of the
differentiation program. In fact, Lamin A/C functions as nucleoplasmic scaffold in both anchoring
chromatin to the nuclear lamina and organizing RNA polymerase II transcriptional complexes
(Dorner et al., 2007; Vlcek and Foisner, 2007; Reddy et al., 2008); as well as Lamin A/C could
interact with some transcription factors (Andres and Gonzalez, 2009). Noteworthy, the expression
of the genes codifying for retinoic acid receptors are not changed in LMNA silenced cells further
supporting our hypothesis.
Extending the analysis to the genes differentially changed in response to retinoic acid we
evidenced a strongly reduced number of down-regulated genes in miRL compared to CTR cells.
Consistent with the inhibition of cell differentiation in miRL cells is the down-regulation of genes
which belong to the
DAVID functional categories such as „nervous system development‟,
„neurogenesis‟, „generation of neurons‟, further confirming that LMNA silencing impair neuronal
differentiation. On the other hand, in CTR cells most of the down-regulated genes belong to
„regulation of cell cycle‟ and „nuclear division‟ categories, confirming that these cells exited from
the cell cycle and acquired the propensity to differentiate. This is also evident analyzing the genes
mainly up-regulated in response to retinoic acid in CTR cells, most of them belonging to the
„nervous system development‟ category.
One of our additional goals was to identify a small subset of genes that are most diagnostic of
sample differences. Analyzing the DAVID functional annotation of the up-regulated genes in miRL
cells exposed to retinoic acid it is evident that the LMNA silenced cells respond to the
differentiating agent by increasing the expression of genes related to tumor progression (e.g. „cell
44
migration‟ and „angiogenesis‟ categories) and not principally to differentiation as one could have
expected giving a differentiation stimulus. Indeed, focusing on the two cell lines not induced to
differentiate we clearly demonstrated the expression of a more aggressive phenotype in the miRL
cells compared to CTR, evidencing the up-regulation of genes related to cancer progression
(metalloproteases, angiogenic factors, and metastases-associated gene) and the down-regulation of
tumor suppressor genes, demonstrating that Lamin A/C contributes to maintain normal cellular
processes. We also demonstrated that alterations of such genes expression resulted in modifications
of some biological behaviors. In fact, consistent with the increased expression of metalloproteases
or metastases associated genes, is an augmented ability of the cells to positively respond to a
migratory stimulus and to increase the secretion of MMP2, with a consequent higher proteolytic
activity of miRL cells. The more aggressive phenotype elicited by the LMNA silenced cells
correlates with a higher resistance to antitumoral chemotherapic treatments. Generally, a more
differentiated tumor phenotype is less aggressive and determines a favorable patient‟s outcome due
to its higher sensitivity to drugs. The greater drug resistance elicited by miRL cells is in part to be
ascribed to the increased expression levels of the multidrug-related efflux pump P-glycoprotein
(Pgp). In fact, the Pgp could be responsible of a reduced intracellular drug amount as indicated by
the decreased intracellular calcein accumulation, widely used as fluorescent probe to assay
membrane permeability (Karaszi et al., 2001). However, no modification in the expression of genes
codifying for Pgp has been observed strongly indicating that a posttranslational event have been
occurred to regulate this protein expression. Indeed, very recent papers reported about the cellular
processes involved in the posttranslational regulation of cellular transport protein such as the ATPbinding cassette Pgp (Yoo et al., 2010) (Titapiwatanakun and Murphy, 2009).
In conclusion, in agreement with other authors (Prokocimer et al., 2006) (Agrelo et al., 2005)
(Stadelmann et al., 1990) (Oguchi et al., 2002) (Venables et al., 2001) (Moss et al., 1999) (Broers et
al., 1993) (Machiels et al., 1997) (Coradeghini et al., 2006), our data clearly demonstrate that low
levels of Lamin A/C expression are associated with poor tumor differentiation and augmented
tumor progression determining a more aggressive phenotype. The characterization of
neuroblastoma tumors on the basis of A-type lamins expression may provide information about the
response of an individual patient‟s tumor to proposed therapy, allowing the development of new
therapeutic strategies based on a rational use of the drugs depending on the molecular profile of the
individual tumor. A-types lamins could represent a good marker of tumor progression and of
sensitiveness to conventional antitumoral therapeutics.
45
6
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57
APPENDIX
58
Table 1. Functional analysis of differential gene lists performed by the DAVID web tool
showing statistically over-represented functional groups in {CTR*-miRL*} .
{CTR*-miRL*} up-regulated genes
Category
Term
Count
%
SP_PIR_KEYWORDS
protein transport
14
5,738
5,79E-03
SNX18
SEC31B
ZMAT3
CCDC91
RAB11FIP2
TOM1L2
BCAP29
AAGAB
CEP290
RAB6B
APPBP2
ERC1
TNPO1
JAKMIP1
GOTERM_BP_4
GO:0006325~chromatin organization
12
4,918
1,27E-02
TSPYL1
CHD9
TBL1XR1
RSF1
HIRA
HDAC10
NAP1L3
JMJD1C
SETD2
BAZ2A
SMARCA2
TCF7L1
GOTERM_BP_4
GO:0007399~nervous system development
23
9,426
PValue Genes
3,29E-02
TWSG1
ADAM23
ALDH5A1
FMR1
CNP
REST
ITM2B
PCDHB10
TIMP3
APLP2
TGFB2
BBS1
NAV2
BAX
SEMA4C
NPTN
CNTN2
CEP290
FOXC1
NRSN1
KCNQ2
CLN8
KALRN
{CTR*-miRL*} down-regulated genes
Category
Term
Count
%
KEGG_PATHWAY
hsa03040:Spliceosome
46
3,061
1,45E-18
CHERP
CCDC12
NHP2L1
TRA2B
U2AF2
SNRPB2
SNRPD1
HSPA1A
SF3B2
CTNNBL1
PRPF19
HSPA1L
HNRNPA3
SF3B1
SFRS7
DDX46
PCBP1
PRPF8
USP39
LSM5
MAGOHB
LSM4
LSM3
SNRNP70
HNRNPC
LSM2
ACIN1
DHX8
EFTUD2
SFRS13A
SFRS1
SF3A2
SF3A1
RBMX
SFRS3
HNRNPU
PRPF6
SNRPB
SNRPA
PHF5A
SNRPF
THOC3
SNRPE
TXNL4A
SNRPG
BAT1
GOTERM_BP_4
GO:0000280~nuclear division
48
3,194
5,37E-10
AURKA
PTTG2
CDCA8
RAD21
OIP5
TARDBP
INCENP
CDCA2
NUP37
CDCA3
ANAPC1
ANAPC2
RAN
SGOL1
UBE2I
UBE2C
DCTN1
NCAPD2
DCLRE1A
CHMP1A
HAUS8
PPP5C
HAUS6
FZR1
CCDC99
HAUS1
TIPIN
ANLN
CEP55
KIAA1009
TUBB
NUMA1
BUB1
PBRM1
BUB3
PINX1
BGLAP
CDC23
CDC20
PMF1
LOC442308
MIS12
CCNB1
FSD1
NOLC1
PLK1
CENPV
SETD8
C13ORF34 TXNL4A
NUP98
NUP160
EIF5A
NUP93
NUP188
NUP214
RAE1
DDX19B
NUP210
NPM1
MAGOHB
NUP37
TPR
NUP35
PABPN1
RANBP17
NUP88
RAN
SFRS13A
SMG1
NUP85
NUP155
DDX39
NUP205
THOC3
TOP3A
GOTERM_BP_4
GO:0050658~RNA transport
26
1,730
PValue Genes
1,40E-07
BAT1
GOTERM_MF_5
GO:0034062~RNA polymerase activity
12
0,798
1,62E-04
POLRMT
POLR2F
POLR3K
POLR2J
POLR2I
POLR3A
POLR1B
POLR2D
MED21
POLR2C
POLR3B
POLR3E
GOTERM_BP_5
GO:0006260~DNA replication
31
2,063
4,15E-04
ING5
HMGB1
NUP98
LIN9
HUS1
NAP1L1
TIPIN
RPA3
TK1
RPA1
TOP1
MCM8
POLE3
POLM
ATRIP
PINX1
CCDC88A
NOL8
CINP
RFC3
ORC3L
RFC1
RRM2
NCOA6
ORC5L
PTMS
RBM14
CHAF1B
DUT
MED1
GOTERM_BP_5
GO:0006281~DNA repair
38
2,528
3,68E-03
RAD23B
HMGB1
RAD51C
HUS1
PTTG2
RPA3
PRPF19
HSPA1L
RPA1
FANCL
FANCM
MUTYH
RAD21
SETMAR
H2AFX
FANCG
ATRIP
NTHL1
RTEL1
WDR33
FANCB
NUDT1
SMG1
XRCC6BP1 RFC3
DCLRE1A
RFC1
TDP1
GADD45G
NCOA6
RAD18
RUVBL2
RBM14
OGG1
PARP2
CHAF1B
BTBD12
BARD1
GOTERM_CC_5
GO:0000792~heterochromatin
10
0,665
4,46E-03
SUZ12
PCGF2
SIN3B
INCENP
TRIM28
H2AFX
MBD3
TOP2B
CBX5
TCP1P3
GOTERM_BP_4
GO:0051726~regulation of cell cycle
41
2,728
7,14E-03
CCNT2
CKS1B
FZR1
ZAK
HUS1
LOC389842
STAT5B
TIPIN
RPS15A
ANLN
BOP1
CASP3
BCL2
NPM1
BUB1
AATF
H2AFX
FANCG
TPR
SIK1
INSR
ATRIP
BUB3
ANAPC2
GTPBP4
GMNN
CDC23
CDKN3
UBE2C
CDK4
PKIA
PTPN11
CCNB1
CDKN1C
PRKCQ
CHMP1A
MAD2L1BP
GADD45G
TSC2
CKS2
RBM38
GOTERM_BP_4
GO:0051253~negative regulation of RNA metabolic process
44
2,927
7,32E-03
HMGB1
HMX1
HAT1
PAX4
GLI2
VPS72
CBX5
RPS26
EPC1
SAP30
PCGF2
SIN3B
NPM1
DDX20
TCF4
SIK1
ENO1
ZNF593
ARID5B
RXRA
RBL1
TRIM28
MLXIPL
SFRS13A
ZNF8
ILF3
UBE2I
MBD3
SPEN
PKIA
SFRS13B
HDAC5
CDKN1C
SUZ12
PA2G4
CHMP1A
SALL4
SMARCE1
JAZF1
RPS13
RBPJ
TBL1X
NSD1
BARD1
HDAC5
SAP30
CSNK2A1
SMARCE1
ACTL6B
MBD3
ARID1B
RBM10
TBL1X
CHD4
CBX5
ANK3
MAP1LC3B
RIBC1
CNN1
ZYX
TUBA1C
CAP2
ACTN4
CCNL2
MED17
C19ORF2
RUVBL1
EP400
GNAO1
ENC1
KIF5C
EPM2A
TP53
GAP43
MYH10
GOTERM_CC_4
GO:0016585~chromatin remodeling complex
11
0,732
5,21E-02
Table 2. Functional analysis of differential gene lists performed by the DAVID web tool
showing statistically over-represented functional groups in {miRL*-CTR*} .
{miRL*-CTR*} up-regulated genes
Category
Term
PIR_SUPERFAMILY
PIRSF002564:metallothionein
GOTERM_BP_5
GO:0006749~glutathione metabolic process
GOTERM_BP_5
GO:0016477~cell migration
PANTHER_MF_ALL
MF00270:Membrane traffic regulatory protein
12
PANTHER_MF_ALL
SP_PIR_KEYWORDS
MF00091:Cytoskeletal protein
angiogenesis
Count
%
5
0,729
5,16E-04
MT2A
MT1B
MT1H
MT1G
MT1X
7
1,020
2,75E-04
G6PD
GPX4
GSTT1
GGT1
GCLM
16
2,332
4,52E-02
PVR
PPARD
SOX1
BCAR1
WASF2
MYH9
GDNF
BTG1
ANG
BAX
CKLF
SDCBP
POU3F3
LAMC1
APBB2
PPAP2A
1,749
5,14E-04
SNX9
LAMP2
PACSIN1
SNX5
STXBP5
SYT12
APBA3
SDCBP
SYNGR2
SYT17
SNX10
SYNGR3
3,48E-03
GFAP
PDLIM7
BCAR1
FKSG2
WASF2
PDLIM4
PDLIM3
RDX
PDLIM1
PROSAPIP1
KIFC3
VCL
TTLL3
KIF13A
KRT81
LOC392288
MC1R
ACTN1
KRT10
DYNLT1
MYL12B
ACTN3
CSRP1
MYL12A
MYH9
KIF1C
FNBP1
TNNT1
SPAG6
TUBA4A
MAP4
WDR1
DNAL1
KATNAL1
TYMP
EGFL7
AGGF1
ANG
EFNA1
ZC3H12A
MFGE8
42
7
6,122
1,020
PValue Genes
2,27E-02
SOD2
GLRX2
{miRL*-CTR*} down-regulated genes
Category
Term
Count
%
GOTERM_CC_4
GO:0016363~nuclear matrix
8
1,839
3,17E-04
GMCL1
FIGN
SFPQ
ENC1
TP53
NUFIP1
RUVBL1
THOC1
GOTERM_CC_4
GO:0044451~nucleoplasm part
27
6,207
PValue Genes
6,54E-04
MEAF6
STK38
SETD1B
NUFIP1
NFYA
IVNS1ABP
COIL
CBX5
SFRS4
HAND2
OGT
GTF3C2
TFDP1
ELP2
RBBP4
TP53
YTHDC1
TOPBP1
SFRS1
USF1
THOC1
MED1
GOTERM_BP_4
GO:0006325~chromatin organization
20
4,598
2,03E-03
MEAF6
RBBP4
RCOR1
SETD1B
CTCF
WHSC1
HLTF
CBX5
EYA3
RPS6KA5
KDM2B
SMARCD2
H2AFV
MSL1
RTF1
SUPT16H
RUVBL1
MLL3
MPHOSPH8
EP400
GOTERM_BP_4
GO:0007399~nervous system development
42
9,655
2,65E-03
FGFR1
NBN
IRX5
CEP120
NNAT
LINGO1
GATA2
GPC2
TUBB
HEY1
CRMP1
SRR
CDK5RAP3
KCNQ2
NRG1
GUSBP3
OLFM1
C17ORF28
TWIST1
IRS2
ALMS1
DPYSL2
PROX1
YWHAE
CTNNA2
ATRX
SHOX2
VEGFA
MTR
RACGAP1P
NAIP
ID4
SEMA4D
RBPJ
GAP43
MED1
MYH10
GOTERM_BP_5
GO:0016568~chromatin modification
16
3,678
3,29E-03
MEAF6
RBBP4
RCOR1
SETD1B
CTCF
WHSC1
HLTF
EYA3
RPS6KA5
KDM2B
SMARCD2
MSL1
RTF1
RUVBL1
MLL3
EP400
GOTERM_BP_4
GO:0022008~neurogenesis
25
5,747
1,06E-02
FGFR1
NBN
CEP120
IRX5
NNAT
GATA2
LINGO1
GPC2
TUBB
CDK5RAP3
C17ORF28
TWIST1
GNAO1
KIF5C
TP53
ALMS1
YWHAE
CTNNA2
VEGFA
RACGAP1P ID4
SEMA4D
RBPJ
GOTERM_BP_5
GO:0006281~DNA repair
15
3,448
1,09E-02
ESCO1
NBN
GEN1
UNG
LIG1
TP53
LIG3
SMG1
TOPBP1
EYA3
ATRX
TYMS
DCLRE1B
SFPQ
SUPT16H
GOTERM_BP_5
GO:0048699~generation of neurons
23
5,287
2,07E-02
FGFR1
NBN
GNAO1
IRX5
NNAT
KIF5C
TP53
ALMS1
YWHAE
CTNNA2
GATA2
LINGO1
GPC2
TUBB
VEGFA
RACGAP1P
CDK5RAP3
ID4
SEMA4D
GAP43
TWIST1
C17ORF28
GOTERM_BP_4
GO:0007420~brain development
14
3,218
2,29E-02
IRS2
CEP120
GNAO1
NNAT
YWHAE
PROX1
CTNNA2
ATRX
GATA2
SRR
ID4
CDK5RAP3
MYH10
MED1
MYH10
Table 3. Differentially expressed genes between CTR and miRL cells
Down-regulated genes in miRL vs CTR cells
Up-regulated genes in miRL vs CTR cells
GENE SYMBOL
Gene Name
GENE SYMBOL
Gene Name
TGFB2
TARSL2
TAGLN
XRCC6BP1
RUNX1T1
CRYGC
POPDC2
CCL24
ZC3HAV1L
PHF2
C20orf141
TIGD7
CSNK1A1L
ATF3
GABPB1
CSNK1G1
CREB3L1
BEX5
CRABP2
HFM1
EIF3A
GALR3
ADM
CHMP4B
AASDH
TMEM38B
IRX5
CHRM2
SKP2
EYA1
SLC35F3
C16orf72
GSC
CSNK1A1P
WNT6
MUC4
LEPRE1
ZNF467
SREBF1
NPM3
CNN1
DLK1
S100A11
TCF4
CDH22
RILPL1
UBXN7
NR4A3
C17orf55
LOC100292283
HIST1H1D
C21orf90
BCAM
RAB3C
GRIN1
MTSS1L
CXXC4
KRTAP2-4
transforming growth factor, beta 2
threonyl-tRNA synthetase-like 2
transgelin
XRCC6 binding protein 1
runt-related transcription factor 1; translocated to, 1 (cyclin D-related)
crystallin, gamma C
popeye domain containing 2
chemokine (C-C motif) ligand 24
zinc finger CCCH-type, antiviral 1-like
PHD finger protein 2
chromosome 20 open reading frame 141
tigger transposable element derived 7
casein kinase 1, alpha 1-like
activating transcription factor 3
GA binding protein transcription factor, beta subunit 1
casein kinase 1, gamma 1
cAMP responsive element binding protein 3-like 1
brain expressed, X-linked 5
cellular retinoic acid binding protein 2
HFM1, ATP-dependent DNA helicase homolog (S. cerevisiae)
eukaryotic translation initiation factor 3, subunit A
galanin receptor 3
adrenomedullin
chromatin modifying protein 4B
aminoadipate-semialdehyde dehydrogenase
transmembrane protein 38B
iroquois homeobox 5
cholinergic receptor, muscarinic 2
S-phase kinase-associated protein 2 (p45)
eyes absent homolog 1 (Drosophila)
solute carrier family 35, member F3
chromosome 16 open reading frame 72
goosecoid homeobox
casein kinase 1, alpha 1 pseudogene
wingless-type MMTV integration site family, member 6
mucin 4, cell surface associated
leucine proline-enriched proteoglycan (leprecan) 1
zinc finger protein 467
sterol regulatory element binding transcription factor 1
nucleophosmin/nucleoplasmin, 3
calponin 1, basic, smooth muscle
delta-like 1 homolog (Drosophila)
S100 calcium binding protein A11; S100 calcium binding protein A11 pseudogene
transcription factor 4
cadherin-like 22
Rab interacting lysosomal protein-like 1
UBX domain protein 7
nuclear receptor subfamily 4, group A, member 3
chromosome 17 open reading frame 55
hypothetical protein LOC100292283
histone cluster 1, H1d
chromosome 21 open reading frame 90
basal cell adhesion molecule (Lutheran blood group)
RAB3C, member RAS oncogene family
glutamate receptor, ionotropic, N-methyl D-aspartate 1
metastasis suppressor 1-like
CXXC finger 4
keratin associated protein 2-1; keratin associated protein 2-4; keratin associated protein 2-3;
similar to keratin associated protein 2-4; keratin associated protein 2-2
splicing factor 3a, subunit 2, 66kDa
prolyl 4-hydroxylase, alpha polypeptide I
retinol binding protein 1, cellular
kinesin family member 1C
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3
basic helix-loop-helix family, member e23
dihydrolipoamide S-succinyltransferase (E2 component of 2-oxo-glutarate complex);
dihydrolipoamide S-succinyltransferase pseudogene (E2 component of 2-oxo-glutarate complex)
neural precursor cell expressed, developmentally down-regulated 4-like
fascin homolog 1, actin-bundling protein (Strongylocentrotus purpuratus)
glutamate receptor, ionotropic, N-methyl D-aspartate 2D
leptin receptor
metallothionein 1A
hypothetical LOC100133660
emerin
myosin, light chain 3, alkali; ventricular, skeletal, slow
family with sequence similarity 100, member A
RNA binding protein with multiple splicing
CCAAT/enhancer binding protein (C/EBP), delta
PX domain containing serine/threonine kinase
KIAA1715
zinc finger protein 700
zinc finger protein 488
mitogen-activated protein kinase kinase kinase 3
forkhead box P1
guanine nucleotide binding protein (G protein), gamma 8
retbindin
connective tissue growth factor
v-mos Moloney murine sarcoma viral oncogene homolog
Ly6/neurotoxin 1
myosin IC
mannosidase, alpha, class 1A, member 2
chromosome 22 open reading frame 39
Rap guanine nucleotide exchange factor (GEF) 5
similar to hCG1742476
protein disulfide isomerase family A, member 2
syncoilin, intermediate filament protein
Rho GTPase activating protein 1
HNF1 homeobox A
hypothetical protein LOC100287521
nuclear factor I/X (CCAAT-binding transcription factor)
hypothetical protein FLJ22184
claudin 11
TNFAIP3 interacting protein 1
WW and C2 domain containing 1
JAZF zinc finger 1
late cornified envelope 3E
zinc finger, matrin type 4
similar to hCG38670
neuralized homolog (Drosophila)
RGM domain family, member A
chromosome 2 open reading frame 14; Uncharacterized protein C2orf14-like 2; Uncharacterized protein C2orf14-like 1
chromosome 5 open reading frame 46
solute carrier family 39 (zinc transporter), member 14
proteasome (prosome, macropain) activator subunit 3 (PA28 gamma; Ki)
poliovirus receptor-related 1 (herpesvirus entry mediator C)
multiple EGF-like-domains 6
TNF receptor-associated factor 3
fragile histidine triad gene
AT rich interactive domain 1B (SWI1-like)
adaptor-related protein complex 1, sigma 1 subunit
insulin-like growth factor 2 (somatomedin A); insulin; INS-IGF2 readthrough transcript
replication factor C (activator 1) 3, 38kDa
splicing factor, arginine/serine-rich 16
coiled-coil domain containing 71
trans-golgi network protein 2
ribosomal protein L22 pseudogene 11; ribosomal protein L22
allograft inflammatory factor 1-like
similar to hCG2023245; hypothetical LOC649294
palladin, cytoskeletal associated protein
olfactory receptor, family 10, subfamily H, member 2
mex-3 homolog D (C. elegans)
microtubule associated serine/threonine kinase family member 4
corticotropin releasing hormone receptor 1
SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily c, member 2
SR-related CTD-associated factor 1
keratin 18; keratin 18 pseudogene 26; keratin 18 pseudogene 19
growth factor, augmenter of liver regeneration
BCL2-antagonist/killer 1; BCL2-like 7 pseudogene 1
solute carrier family 25, member 29
B-cell CLL/lymphoma 11A (zinc finger protein)
pyrophosphatase (inorganic) 2
similar to TRIMCyp
protein tyrosine phosphatase, non-receptor type 1
KIAA1305
G protein-coupled receptor kinase 5
multiple EGF-like-domains 8
limb bud and heart development homolog (mouse)
chromosome 15 open reading frame 39
regulation of nuclear pre-mRNA domain containing 1A
cell division cycle associated 7-like
hypothetical LOC441245
poly (ADP-ribose) polymerase family, member 10
non-protein coding RNA 85
even-skipped homeobox 1
transducin-like enhancer of split 3 (E(sp1) homolog, Drosophila)
odz, odd Oz/ten-m homolog 3 (Drosophila)
proline rich 7 (synaptic)
receptor (G protein-coupled) activity modifying protein 1
forkhead box Q1
MAS-related GPR, member F
endoplasmic reticulum-golgi intermediate compartment (ERGIC) 1
zinc finger protein 331
late cornified envelope 2D
caldesmon 1
engrailed homeobox 2
similar to Pyruvate kinase, isozymes M1/M2 (Pyruvate kinase muscle isozyme) (Cytosolic thyroid hormone-binding protein)
(CTHBP) (THBP1); pyruvate kinase, muscle
chromosome 6 open reading frame 176
myeloid/lymphoid or mixed-lineage leukemia 3
translocator protein (18kDa)
chromosome 1 open reading frame 113
potassium large conductance calcium-activated channel, subfamily M, beta member 1
microtubule-associated protein 1 light chain 3 alpha
apoptosis inhibitor
signal transducer and activator of transcription 5B
otoferlin
hypothetical protein LOC100129034
hect domain and RLD 5
mindbomb homolog 1 (Drosophila)
potassium voltage-gated channel, subfamily G, member 1
choline phosphotransferase 1
coiled-coil domain containing 109B
glycolipid transfer protein; glycolipid transfer protein pseudogene 1
hypothetical LOC728875
CCAAT/enhancer binding protein (C/EBP), alpha
hypothetical LOC255031
protein kinase C, eta
chromosome 17 open reading frame 96
hypothetical gene supported by AF216292; NM_005347; heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa)
similar to ADAMTS-like 2; ADAMTS-like 2
ACTB pseudogene
upstream binding transcription factor, RNA polymerase I
phosphodiesterase 4B, cAMP-specific (phosphodiesterase E4 dunce homolog, Drosophila)
naked cuticle homolog 2 (Drosophila)
BCL2 binding component 3
potassium voltage-gated channel, Shaw-related subfamily, member 3
similar to cytochrome c oxidase subunit II
zinc finger protein 1 homolog (mouse)
hypothetical LOC100126784
brain protein 44-like
SLIT-ROBO Rho GTPase activating protein 1
glial fibrillary acidic protein
stimulator of chondrogenesis 1
cytokine receptor-like factor 1
dystrophia myotonica-protein kinase
ankyrin repeat domain 2 (stretch responsive muscle)
SERTA domain containing 4
UDP glycosyltransferase 8
chromosome 12 open reading frame 51
coiled-coil domain containing 50
chromosome 16 open reading frame 14
DnaJ (Hsp40) homolog, subfamily C, member 18
inositol 1,4,5-triphosphate receptor interacting protein-like 1
polymerase (DNA directed), mu
ring finger protein 113B
transducin (beta)-like 1X-linked
metallothionein 1G
hypothetical LOC541472
ribonucleoprotein, PTB-binding 2
carbohydrate (chondroitin 4) sulfotransferase 13
Cas-Br-M (murine) ecotropic retroviral transforming sequence
KIAA1199
mitochondrial protein 18 kDa
methyl CpG binding protein 2 (Rett syndrome)
LIM domain only 4
acidic (leucine-rich) nuclear phosphoprotein 32 family, member C
mediator complex subunit 1
adenomatosis polyposis coli 2
chromosome 14 open reading frame 43
aldolase A, fructose-bisphosphate pseudogene 2
ubiquilin 1
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 3, 9kDa
retinoic acid receptor, alpha
pirin (iron-binding nuclear protein)
protein phosphatase methylesterase 1
similar to hCG1991431; similar to COMPase; ADAM metallopeptidase with thrombospondin type 1 motif, 7
similar to hCG2004312
adrenergic, alpha-2A-, receptor
malignant fibrous histiocytoma amplified sequence 1
nucleotide binding protein-like
metallothionein 1L (gene/pseudogene); metallothionein 1E; metallothionein 1 pseudogene 3; metallothionein 1J (pseudogene)
Josephin domain containing 2
ethylmalonic encephalopathy 1
GLI family zinc finger 2
lamin B2
SH3 domain containing 20 pseudogene
metallothionein 2A
ADAM metallopeptidase with thrombospondin type 1 motif, 5
silver homolog (mouse)
similar to AAA-ATPase TOB3; ATPase family, AAA domain containing 3B
hypothetical protein FLJ40504
sema domain, seven thrombospondin repeats (type 1 and type 1-like),
transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B
5,10-methylenetetrahydrofolate reductase (NADPH)
ER lipid raft associated 2
dynein, cytoplasmic 1, light intermediate chain 2
collagen, type V, alpha 2
salt-inducible kinase 2
latent transforming growth factor beta binding protein 1
sidekick homolog 2 (chicken)
neurogenin 3
tetraspanin 10
huntingtin interacting protein 1
centrosomal protein 250kDa
transcription factor AP-2 beta (activating enhancer binding protein 2 beta)
synapsin I
cytoskeleton associated protein 2-like
chromosome 19 open reading frame 73
aquaporin 2 (collecting duct)
phosphoglycerate kinase 1
MAP/microtubule affinity-regulating kinase 2
DNM1 pseudogene 35
CREB binding protein
unc-119 homolog (C. elegans)
phosphotyrosine interaction domain containing 1
immunoglobulin superfamily, member 1
PDZ domain containing 4
ubiquitin specific peptidase 48
parathymosin
peroxisomal biogenesis factor 6
SP100 nuclear antigen
histone cluster 1, H4l; histone cluster 1, H4k; histone cluster 4, H4; histone cluster 1, H4h; histone cluster 1, H4j; histone cluster 1, H4i; histone cluster 1, H4d;
histone cluster 1, H4c; histone cluster 1, H4f; histone cluster 1, H4e; histone cluster 1, H4b; histone cluster 1, H4a; histone cluster 2, H4a; histone cluster 2, H4b
histone cluster 1, H4l; histone cluster 1, H4k; histone cluster 4, H4; histone cluster 1, H4h; histone cluster 1, H4j; histone cluster 1, H4i; histone cluster 1, H4d;
histone cluster 1, H4c; histone cluster 1, H4f; histone cluster 1, H4e; histone cluster 1, H4b; histone cluster 1, H4a; histone cluster 2, H4a; histone cluster 2, H4b
histone cluster 1, H4l; histone cluster 1, H4k; histone cluster 4, H4; histone cluster 1, H4h; histone cluster 1, H4j; histone cluster 1, H4i; histone cluster 1, H4d;
histone cluster 1, H4c; histone cluster 1, H4f; histone cluster 1, H4e; histone cluster 1, H4b; histone cluster 1, H4a; histone cluster 2, H4a; histone cluster 2, H4b
egl nine homolog 1 (C. elegans)
leucine rich repeat containing 28
fibronectin 1
zinc finger protein 205
sorbin and SH3 domain containing 3
family with sequence similarity 123B
hypothetical protein LOC388564
polycystic kidney disease 2 (autosomal dominant)
glial cell derived neurotrophic factor
keratin associated protein 4-1
dihydropyrimidinase-like 3
late cornified envelope 1D
3-phosphoinositide dependent protein kinase-1
GATA zinc finger domain containing 1
l(3)mbt-like 3 (Drosophila)
Nance-Horan syndrome (congenital cataracts and dental anomalies)
transmembrane protein 175
cholinergic receptor, muscarinic 3
histone cluster 1, H3j; histone cluster 1, H3i; histone cluster 1, H3h; histone cluster 1, H3g; histone cluster 1, H3f; histone cluster 1, H3e; histone cluster 1, H3d;
histone cluster 1, H3c; histone cluster 1, H3b; histone cluster 1, H3a; histone cluster 1, H2ad; histone cluster 2, H3a; histone cluster 2, H3c; histone cluster 2, H3d
histone cluster 1, H3j; histone cluster 1, H3i; histone cluster 1, H3h; histone cluster 1, H3g; histone cluster 1, H3f; histone cluster 1, H3e; histone cluster 1, H3d;
histone cluster 1, H3c; histone cluster 1, H3b; histone cluster 1, H3a; histone cluster 1, H2ad; histone cluster 2, H3a; histone cluster 2, H3c; histone cluster 2, H3d
histone cluster 1, H3j; histone cluster 1, H3i; histone cluster 1, H3h; histone cluster 1, H3g; histone cluster 1, H3f; histone cluster 1, H3e; histone cluster 1, H3d;
histone cluster 1, H3c; histone cluster 1, H3b; histone cluster 1, H3a; histone cluster 1, H2ad; histone cluster 2, H3a; histone cluster 2, H3c; histone cluster 2, H3d
histone cluster 1, H3j; histone cluster 1, H3i; histone cluster 1, H3h; histone cluster 1, H3g; histone cluster 1, H3f; histone cluster 1, H3e; histone cluster 1, H3d;
histone cluster 1, H3c; histone cluster 1, H3b; histone cluster 1, H3a; histone cluster 1, H2ad; histone cluster 2, H3a; histone cluster 2, H3c; histone cluster 2, H3d
interleukin 27
carcinoembryonic antigen-related cell adhesion molecule 20
zinc finger protein 575
sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1
c-Maf-inducing protein
hypothetical protein LOC728449
zinc finger protein 433
microtubule-associated protein, RP/EB family, member 2
heme binding protein 2
similar to keratin 18
additional sex combs like 3 (Drosophila)
transmembrane protein 88
actinin, alpha 4
interferon induced transmembrane protein 5
SRY (sex determining region Y)-box 3
striatin, calmodulin binding protein 4
leukocyte receptor cluster (LRC) member 8
sterile alpha motif domain containing 4A
mitochondrial ribosomal protein L23
centromere protein N
ADAM metallopeptidase domain 15
serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2
aquaporin 1 (Colton blood group)
lamin A/C
v-akt murine thymoma viral oncogene homolog 2
heparin-binding EGF-like growth factor
mitochondrial ribosomal protein S36
cadherin-like 24
tet oncogene 1
tryptase gamma 1
hyperpolarization activated cyclic nucleotide-gated potassium channel 4
fibronectin leucine rich transmembrane protein 3
annexin A6
protein kinase N2
CREB regulated transcription coactivator 1
sorbin and SH3 domain containing 2
ribosomal protein S2 pseudogene 45
PDZ and LIM domain 5
POM121 membrane glycoprotein (rat)
extracellular leucine-rich repeat and fibronectin type III domain containing 1
chromosome 6 open reading frame 27
hypothetical protein LOC283711
inhibin, beta A
urotensin 2 receptor
PARK2 co-regulated
hypothetical gene supported by BC000665; t-complex 1
ribosomal protein L17 pseudogene 22; ribosomal protein L17 pseudogene 36; ribosomal protein L17 pseudogene 20; similar to ribosomal protein L17;
ribosomal protein L17 pseudogene 33; ribosomal protein L17 pseudogene 34; ribosomal protein L17 pseudogene 9; ribosomal protein L17;
ribosomal protein L17 pseudogene 18; ribosomal protein L17 pseudogene 7; ribosomal protein L17 pseudogene 39
transforming growth factor, beta 3
BOD1L
FAM20C
SSTR2
CNTNAP2
AK5
MSX2
KIDINS220
SPON2
SYNPO
RAB11FIP2
GREM1
MALAT1
ZNF397
STK19
ADAM23
TM4SF1
BAALC
HS6ST3
P4HTM
PIP5K1A
GALNT14
SCG2
TMEM204
TNIP2
HS3ST5
HMOX1
BTBD11
PSD3
CD86
CADM3
GPR68
KIAA1033
REST
HIRA
RSC1A1
PTPRE
KCTD12
ETV5
TUBA1C
C3orf62
AHNAK
ZFX
ARHGEF7
IGFBP5
ACP2
HTR2B
MARVELD1
PRKAG1
PCDH17
XPR1
SCG5
PROCR
PURG
OAZ3
FAP
C1orf96
C9orf129
APOE
HNRNPU
DKK2
CHST8
JMJD1C
NGFR
CTHRC1
EEF1D
CADPS
TAOK1
GRIA3
S100A16
TOM1L2
BAZ2A
TNPO1
ANKRD32
RALGDS
EML2
GREM2
ENPP2
CLDN5
MMP15
MSN
IRS2
NRXN3
PPP1R8
LRRC17
PRPH
TRIB2
PPP2R2B
PHLDA1
LOC100132831
PPP2R2C
GFRA2
ASB8
OTUD4
RAPGEF4
PRR13
AAGAB
PLAU
MAOB
H6PD
FGL1
biorientation of chromosomes in cell division 1-like
family with sequence similarity 20, member C
somatostatin receptor 2
contactin associated protein-like 2
adenylate kinase 5
msh homeobox 2
kinase D-interacting substrate, 220kDa
spondin 2, extracellular matrix protein
synaptopodin
RAB11 family interacting protein 2 (class I)
gremlin 1, cysteine knot superfamily, homolog (Xenopus laevis)
metastasis associated lung adenocarcinoma transcript 1 (non-protein coding)
zinc finger protein 397
serine/threonine kinase 19
ADAM metallopeptidase domain 23
transmembrane 4 L six family member 1
brain and acute leukemia, cytoplasmic
heparan sulfate 6-O-sulfotransferase 3
prolyl 4-hydroxylase, transmembrane (endoplasmic reticulum)
phosphatidylinositol-4-phosphate 5-kinase, type I, alpha
UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 14 (GalNAc-T14)
secretogranin II (chromogranin C)
transmembrane protein 204
TNFAIP3 interacting protein 2
heparan sulfate (glucosamine) 3-O-sulfotransferase 5
heme oxygenase (decycling) 1
BTB (POZ) domain containing 11
pleckstrin and Sec7 domain containing 3
CD86 molecule
cell adhesion molecule 3
G protein-coupled receptor 68
KIAA1033
RE1-silencing transcription factor
HIR histone cell cycle regulation defective homolog A (S. cerevisiae)
regulatory solute carrier protein, family 1, member 1
protein tyrosine phosphatase, receptor type, E
potassium channel tetramerisation domain containing 12
ets variant 5
tubulin, alpha 1c
chromosome 3 open reading frame 62
AHNAK nucleoprotein
zinc finger protein, X-linked
Rho guanine nucleotide exchange factor (GEF) 7
insulin-like growth factor binding protein 5
acid phosphatase 2, lysosomal
5-hydroxytryptamine (serotonin) receptor 2B
hypothetical LOC100270710; MARVEL domain containing 1
protein kinase, AMP-activated, gamma 1 non-catalytic subunit
protocadherin 17
xenotropic and polytropic retrovirus receptor
secretogranin V (7B2 protein)
protein C receptor, endothelial (EPCR)
purine-rich element binding protein G
ornithine decarboxylase antizyme 3
fibroblast activation protein, alpha
chromosome 1 open reading frame 96
chromosome 9 open reading frame 129
hypothetical LOC100129500; apolipoprotein E
heterogeneous nuclear ribonucleoprotein U (scaffold attachment factor A)
dickkopf homolog 2 (Xenopus laevis)
carbohydrate (N-acetylgalactosamine 4-0) sulfotransferase 8
jumonji domain containing 1C
nerve growth factor receptor (TNFR superfamily, member 16)
collagen triple helix repeat containing 1
eukaryotic translation elongation factor 1 delta (guanine nucleotide exchange protein)
Ca++-dependent secretion activator
TAO kinase 1
glutamate receptor, ionotrophic, AMPA 3
S100 calcium binding protein A16
target of myb1-like 2 (chicken)
bromodomain adjacent to zinc finger domain, 2A
transportin 1
ankyrin repeat domain 32
ral guanine nucleotide dissociation stimulator
echinoderm microtubule associated protein like 2
gremlin 2, cysteine knot superfamily, homolog (Xenopus laevis)
ectonucleotide pyrophosphatase/phosphodiesterase 2
claudin 5
matrix metallopeptidase 15 (membrane-inserted)
moesin
insulin receptor substrate 2
neurexin 3
protein phosphatase 1, regulatory (inhibitor) subunit 8
leucine rich repeat containing 17
peripherin
tribbles homolog 2 (Drosophila)
protein phosphatase 2 (formerly 2A), regulatory subunit B, beta isoform
pleckstrin homology-like domain, family A, member 1
A20-binding inhibitor of NF-kappaB activation 2 pseudogene
protein phosphatase 2 (formerly 2A), regulatory subunit B, gamma isoform
similar to GDNF family receptor alpha 2; GDNF family receptor alpha 2
ankyrin repeat and SOCS box-containing 8
OTU domain containing 4
Rap guanine nucleotide exchange factor (GEF) 4
proline rich 13
alpha- and gamma-adaptin-binding protein p34
plasminogen activator, urokinase
monoamine oxidase B
hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase)
fibrinogen-like 1
SF3A2
P4HA1
RBP1
KIF1C
PFKFB3
BHLHE23
DLST
NEDD4L
FSCN1
GRIN2D
LEPR
MT1A
LOC100133660
EMD
MYL3
FAM100A
RBPMS
CEBPD
PXK
KIAA1715
ZNF700
ZNF488
MAP3K3
FOXP1
GNG8
RTBDN
CTGF
MOS
LYNX1
MYO1C
MAN1A2
C22orf39
RAPGEF5
LOC402360
PDIA2
SYNC
ARHGAP1
HNF1A
LOC100287521
NFIX
FLJ22184
CLDN11
TNIP1
WWC1
JAZF1
LCE3E
ZMAT4
LOC100132644
NEURL
RGMA
C2orf14
C5orf46
SLC39A14
PSME3
PVRL1
MEGF6
TRAF3
FHIT
ARID1B
AP1S1
IGF2
RFC3
SFRS16
CCDC71
TGOLN2
RPL22
AIF1L
LOC649294
PALLD
OR10H2
MEX3D
MAST4
CRHR1
SMARCC2
SCAF1
KRT18
GFER
BAK1
SLC25A29
BCL11A
PPA2
LOC392352
PTPN1
NYNRIN
GRK5
MEGF8
LBH
C15orf39
RPRD1A
CDCA7L
LOC441245
PARP10
NCRNA00085
EVX1
TLE3
ODZ3
PRR7
RAMP1
FOXQ1
MRGPRF
ERGIC1
ZNF331
LCE2D
CALD1
EN2
PKM2
C6orf176
MLL3
TSPO
C1orf113
KCNMB1
MAP1LC3A
FKSG2
STAT5B
OTOF
LOC100129034
HERC5
MIB1
KCNG1
CHPT1
CCDC109B
GLTP
LOC728875
CEBPA
FLJ35390
PRKCH
C17orf96
HSPA5
ADAMTSL2
LOC648740
UBTF
PDE4B
NKD2
BBC3
KCNC3
LOC100288578
ZFP1
LOC100126784
BRP44L
SRGAP1
GFAP
SCRG1
CRLF1
DMPK
ANKRD2
SERTAD4
UGT8
C12orf51
CCDC50
FAM195A
DNAJC18
ITPRIPL1
POLM
RNF113B
TBL1X
MT1G
LOC541472
RAVER2
CHST13
CBL
KIAA1199
MTP18
MECP2
LMO4
ANP32C
MED1
APC2
C14orf43
ALDOAP2
UBQLN1
NDUFA3
RARA
PIR
PPME1
ADAMTS7
LOC100288367
ADRA2A
MFHAS1
NUBPL
MT1L
JOSD2
ETHE1
GLI2
LMNB2
LOC440461
MT2A
ADAMTS5
SILV
ATAD3B
FLJ40504
SEMA5B
MTHFR
ERLIN2
DYNC1LI2
COL5A2
SIK2
LTBP1
SDK2
NEUROG3
TSPAN10
HIP1
CEP250
TFAP2B
SYN1
CKAP2L
C19orf73
AQP2
PGK1
MARK2
DNM1P35
CREBBP
UNC119
PID1
IGSF1
PDZD4
USP48
PTMS
PEX6
SP100
HIST1H4B
HIST1H4D
HIST1H4L
EGLN1
LRRC28
FN1
ZNF205
SORBS3
FAM123B
LOC388564
PKD2
GDNF
KRTAP4-1
DPYSL3
LCE1D
PDPK1
GATAD1
L3MBTL3
NHS
TMEM175
CHRM3
HIST1H3J
HIST1H3D
HIST1H3B
HIST1H3F
IL27
CEACAM20
ZNF575
SPOCK1
CMIP
LOC728449
ZNF433
MAPRE2
HEBP2
LOC442249
ASXL3
TMEM88
ACTN4
IFITM5
SOX3
STRN4
LENG8
SAMD4A
MRPL23
CENPN
ADAM15
SERPINE2
AQP1
LMNA
AKT2
HBEGF
MRPS36
CDH24
TET1
TPSG1
HCN4
FLRT3
ANXA6
PKN2
CRTC1
SORBS2
RPS2P45
PDLIM5
POM121
ELFN1
C6orf27
LOC283711
INHBA
UTS2R
PACRG
TCP1P3
LOC729046
TGFB3
TSHZ1
C14orf102
PRR5
TMBIM6
TSPAN12
DBH
HES7
HRK
NPPB
LRRN1
DKK1
CYB5R3
RPL22P15
MLEC
LOC645181
PDAP1
LOC100130152
CHD3
MAPK11
ING5
RPL35
COL27A1
BNIP3
SPEN
GNG5
MSI2
GTPBP1
DIAPH2
TSEN15
PRNP
SCOC
PITPNM1
ANG
ZNF236
MDK
CDC42EP5
DCN
TNRC18
SYK
MRPL2
CACNG7
PRR12
PDK1
HCN2
RPL23AP32
FAM43B
MT1X
LOC100131581
EDN1
CAB39L
KIT
H19
RDH11
KIF1B
FKBP10
KLF10
TSPAN6
ZCCHC2
RND3
RHBDL1
GPT2
LOC440917
KISS1R
COL16A1
NOB1
NUFIP1
POLR2C
PSMB9
SPRY2
SLC27A2
LOC642031
WASF2
PRPF19
FBXL17
CD4
RCOR3
POU3F3
ZC3H7B
MYPOP
C18orf23
UBE2W
HS2ST1
VASH2
RARG
TWIST2
PFKFB4
ALDOA
ZSCAN10
LOC152024
C8orf4
PPAP2B
PPP2R3B
EYA4
RHOG
MYH14
RALA
GATC
MT3
FTL
GPC4
DDR2
FCRLB
GPRIN1
HK2
TRIM25
FAM59A
LRP5
SPPL2B
CASP2
LOC646973
TAF3
ZNF579
PTCH1
FAM124A
PDLIM3
TIFA
ANXA5
PBX2
CACNG4
LOC100128164
ACTN3
COX8A
FMNL2
AGAP3
VGF
CACNA1I
LCE1A
PXMP4
CAND1
ITPKB
FAM133B
LRRC8A
FILIP1L
IER5
MT1H
THRAP3
CASKIN1
CAPG
ING3
COX6A2
MALAT1
HRASLS
C3orf32
BGLAP
TESC
SLC2A13
EXPH5
NBEAL2
TAF15
SLC1A1
LPP
FANCB
DPP9
LOH3CR2A
LOC100288755
CDK5R2
RNF5
CDK6
C14orf21
ZNF48
SLIT1
BCL2L12
GPRC5B
OR4D2
RPS26
NTNG2
SRC
NDOR1
ZNF234
DLGAP3
C9orf69
NDUFS7
AATK
IRX3
KIAA0319L
ELN
EPDR1
PLK1S1
WDR4
LOC100289410
INSR
LOC650226
GOLPH3
CCDC108
NEDD9
SYCE1L
PRR24
KLK3
NAV2
CTRB2
DIAPH1
C16orf68
LOC100129122
C17orf74
LBX1
DIRC1
PRSS36
BMPR1B
LCE5A
CYB561D1
TTYH1
CLMN
NACAP1
LOC100291560
PMEPA1
ARHGDIA
DHRS2
PPP3R1
TBC1D2B
CCND1
LRP6
AAAS
PIGL
ZSCAN5A
SLC8A2
OR2H1
ALPPL2
UQCRC1
PRKACA
LOC100292388
CCDC124
TPI1
DHDH
PRIC285
RAB6B
C14orf139
HSPB1
STAC2
FAM162A
CYP4V2
EXOC3L2
PIP5K1C
CAMK1D
NPAS3
ARC
LCE1C
ATF6B
CHDH
FTSJ3
ZNF385A
DOCK7
CARHSP1
PHF20
MAGEE1
SNX10
SH3KBP1
C16orf81
FAM129B
PABPC4L
MCAT
TRIM44
CBLL1
KCNC1
C21orf58
RTN3
CSTF2T
ZNF124
CD99
TMTC2
TPR
TMEM99
CPSF1
SLC38A7
TMEM101
TES
IQSEC2
ADD1
AK3L1
ACTG2
TAS1R1
GOLGA8F
LOC100132161
LMNB1
EXOC5
RAB5C
TSPAN13
CD151
EPHX3
MT1B
C11orf75
RAPGEF1
APH1A
SOX2
LOC100131149
LOC728855
ARHGEF16
teashirt zinc finger homeobox 1
chromosome 14 open reading frame 102
Rho GTPase activating protein 8; proline rich 5 (renal); PRR5-ARHGAP8 fusion
transmembrane BAX inhibitor motif containing 6
tetraspanin 12
dopamine beta-hydroxylase (dopamine beta-monooxygenase)
hairy and enhancer of split 7 (Drosophila)
harakiri, BCL2 interacting protein (contains only BH3 domain)
natriuretic peptide precursor B
leucine rich repeat neuronal 1
dickkopf homolog 1 (Xenopus laevis)
cytochrome b5 reductase 3
ribosomal protein L22 pseudogene 15
malectin
PDGFA associated protein 1; similar to PDGFA associated protein 1
PDGFA associated protein 1; similar to PDGFA associated protein 1
hypothetical protein LOC100130152
chromodomain helicase DNA binding protein 3
mitogen-activated protein kinase 11
inhibitor of growth family, member 5
ribosomal protein L35; ribosomal protein L35 pseudogene 1; ribosomal protein L35 pseudogene 2
collagen, type XXVII, alpha 1
BCL2/adenovirus E1B 19kDa interacting protein 3
spen homolog, transcriptional regulator (Drosophila)
guanine nucleotide binding protein (G protein), gamma 5
musashi homolog 2 (Drosophila)
GTP binding protein 1
diaphanous homolog 2 (Drosophila)
tRNA splicing endonuclease 15 homolog (S. cerevisiae)
prion protein
short coiled-coil protein
phosphatidylinositol transfer protein, membrane-associated 1
angiogenin, ribonuclease, RNase A family, 5
zinc finger protein 236
midkine (neurite growth-promoting factor 2)
CDC42 effector protein (Rho GTPase binding) 5
decorin
trinucleotide repeat containing 18
spleen tyrosine kinase
mitochondrial ribosomal protein L2
calcium channel, voltage-dependent, gamma subunit 7
proline rich 12
pyruvate dehydrogenase kinase, isozyme 1
hyperpolarization activated cyclic nucleotide-gated potassium channel 2
ribosomal protein L23a pseudogene 32
family with sequence similarity 43, member B
metallothionein 1X
hypothetical LOC100131581
endothelin 1
calcium binding protein 39-like
similar to Mast/stem cell growth factor receptor precursor (SCFR) (Proto-oncogene tyrosine-protein kinase Kit) (c-kit) (CD117 antigen);
v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog
H19, imprinted maternally expressed transcript (non-protein coding)
retinol dehydrogenase 11 (all-trans/9-cis/11-cis)
kinesin family member 1B
FK506 binding protein 10, 65 kDa
Kruppel-like factor 10
tetraspanin 6
zinc finger, CCHC domain containing 2
Rho family GTPase 3
rhomboid, veinlet-like 1 (Drosophila)
glutamic pyruvate transaminase (alanine aminotransferase) 2
similar to 14-3-3 protein epsilon (14-3-3E) (Mitochondrial import stimulation factor L subunit) (MSF L);
tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide
KISS1 receptor
collagen, type XVI, alpha 1
NIN1/RPN12 binding protein 1 homolog (S. cerevisiae); hypothetical LOC100132364
nuclear fragile X mental retardation protein interacting protein 1
polymerase (RNA) II (DNA directed) polypeptide C, 33kDa
proteasome (prosome, macropain) subunit, beta type, 9 (large multifunctional peptidase 2)
sprouty homolog 2 (Drosophila)
solute carrier family 27 (fatty acid transporter), member 2
hypothetical protein LOC642031
WAS protein family, member 2
PRP19/PSO4 pre-mRNA processing factor 19 homolog (S. cerevisiae)
F-box and leucine-rich repeat protein 17
CD4 molecule
REST corepressor 3
POU class 3 homeobox 3
zinc finger CCCH-type containing 7B
Myb-related transcription factor, partner of profilin
chromosome 18 open reading frame 23
ubiquitin-conjugating enzyme E2W (putative)
heparan sulfate 2-O-sulfotransferase 1
vasohibin 2
retinoic acid receptor, gamma
twist homolog 2 (Drosophila)
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4
aldolase A, fructose-bisphosphate
zinc finger and SCAN domain containing 10
hypothetical LOC152024
chromosome 8 open reading frame 4
phosphatidic acid phosphatase type 2B
protein phosphatase 2 (formerly 2A), regulatory subunit B'', beta
eyes absent homolog 4 (Drosophila)
ras homolog gene family, member G (rho G)
myosin, heavy chain 14
v-ral simian leukemia viral oncogene homolog A (ras related)
glutamyl-tRNA(Gln) amidotransferase, subunit C homolog (bacterial)
metallothionein 3
similar to ferritin, light polypeptide; ferritin, light polypeptide
glypican 4
discoidin domain receptor tyrosine kinase 2
Fc receptor-like B
G protein regulated inducer of neurite outgrowth 1
hexokinase 2 pseudogene; hexokinase 2
tripartite motif-containing 25
hypothetical protein LOC100130616; family with sequence similarity 59, member A
low density lipoprotein receptor-related protein 5
signal peptide peptidase-like 2B
caspase 2, apoptosis-related cysteine peptidase
hypothetical LOC646973
TAF3 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 140kDa
zinc finger protein 579
patched homolog 1 (Drosophila)
family with sequence similarity 124A
PDZ and LIM domain 3
TRAF-interacting protein with forkhead-associated domain
annexin A5
pre-B-cell leukemia homeobox 2
calcium channel, voltage-dependent, gamma subunit 4
four and a half LIM domains 1 pseudogene
actinin, alpha 3
cytochrome c oxidase subunit 8A (ubiquitous)
formin-like 2
ArfGAP with GTPase domain, ankyrin repeat and PH domain 3
VGF nerve growth factor inducible
calcium channel, voltage-dependent, T type, alpha 1I subunit
late cornified envelope 1A
peroxisomal membrane protein 4, 24kDa
cullin-associated and neddylation-dissociated 1
inositol 1,4,5-trisphosphate 3-kinase B
family with sequence similarity 133, member B pseudogene; similar to FAM133B protein; family with sequence similarity 133, member B
leucine rich repeat containing 8 family, member A
filamin A interacting protein 1-like
immediate early response 5
metallothionein 1H
thyroid hormone receptor associated protein 3
CASK interacting protein 1
capping protein (actin filament), gelsolin-like
inhibitor of growth family, member 3
cytochrome c oxidase subunit VIa polypeptide 2
metastasis associated lung adenocarcinoma transcript 1 (non-protein coding)
HRAS-like suppressor
chromosome 3 open reading frame 32
bone gamma-carboxyglutamate (gla) protein; polyamine-modulated factor 1
tescalcin
solute carrier family 2 (facilitated glucose transporter), member 13
exophilin 5
neurobeachin-like 2
TAF15 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 68kDa
solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, system Xag), member 1
LIM domain containing preferred translocation partner in lipoma
Fanconi anemia, complementation group B
dipeptidyl-peptidase 9
loss of heterozygosity, 3, chromosomal region 2, gene A
hypothetical LOC100288755
cyclin-dependent kinase 5, regulatory subunit 2 (p39)
ring finger protein 5; ring finger protein 5 pseudogene 1
cyclin-dependent kinase 6
chromosome 14 open reading frame 21
zinc finger protein 48
slit homolog 1 (Drosophila)
BCL2-like 12 (proline rich)
G protein-coupled receptor, family C, group 5, member B
olfactory receptor, family 4, subfamily D, member 2
ribosomal protein S26 pseudogene 38; ribosomal protein S26 pseudogene 39; ribosomal protein S26 pseudogene 35; ribosomal protein S26 pseudogene 31;
ribosomal protein S26 pseudogene 20; ribosomal protein S26 pseudogene 54; ribosomal protein S26 pseudogene 2; ribosomal protein S26 pseudogene 53;
ribosomal protein S26 pseudogene 25; ribosomal protein S26 pseudogene 50; ribosomal protein S26 pseudogene 6;
ribosomal protein S26 pseudogene 8; ribosomal protein S26
netrin G2
v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian)
NADPH dependent diflavin oxidoreductase 1
zinc finger protein 234
discs, large (Drosophila) homolog-associated protein 3
chromosome 9 open reading frame 69
NADH dehydrogenase (ubiquinone) Fe-S protein 7, 20kDa (NADH-coenzyme Q reductase)
apoptosis-associated tyrosine kinase
iroquois homeobox 3
KIAA0319-like
elastin
ependymin related protein 1 (zebrafish)
non-protein coding RNA 153
WD repeat domain 4
similar to hCG1774568
insulin receptor
ankyrin repeat domain 26 pseudogene
golgi phosphoprotein 3 (coat-protein)
coiled-coil domain containing 108
neural precursor cell expressed, developmentally down-regulated 9
hypothetical protein LOC100130958
hypothetical protein LOC255783
kallikrein-related peptidase 3
neuron navigator 2
chymotrypsinogen B2
diaphanous homolog 1 (Drosophila)
chromosome 16 open reading frame 68
hypothetical LOC100129122
chromosome 17 open reading frame 74
ladybird homeobox 1
disrupted in renal carcinoma 1
protease, serine, 36
bone morphogenetic protein receptor, type IB
late cornified envelope 5A
cytochrome b-561 domain containing 1
tweety homolog 1 (Drosophila)
calmin (calponin-like, transmembrane)
nascent-polypeptide-associated complex alpha polypeptide pseudogene 1
hypothetical protein LOC100291560
prostate transmembrane protein, androgen induced 1
Rho GDP dissociation inhibitor (GDI) alpha
dehydrogenase/reductase (SDR family) member 2
protein phosphatase 3 (formerly 2B), regulatory subunit B, alpha isoform
TBC1 domain family, member 2B
cyclin D1
low density lipoprotein receptor-related protein 6
achalasia, adrenocortical insufficiency, alacrimia (Allgrove, triple-A)
phosphatidylinositol glycan anchor biosynthesis, class L
zinc finger and SCAN domain containing 5A
solute carrier family 8 (sodium/calcium exchanger), member 2
olfactory receptor, family 2, subfamily H, member 1
alkaline phosphatase, placental-like 2
ubiquinol-cytochrome c reductase core protein I
protein kinase, cAMP-dependent, catalytic, alpha
hypothetical protein LOC100292388
coiled-coil domain containing 124
TPI1 pseudogene; triosephosphate isomerase 1
dihydrodiol dehydrogenase (dimeric)
peroxisomal proliferator-activated receptor A interacting complex 285
RAB6B, member RAS oncogene family
chromosome 14 open reading frame 139
heat shock 27kDa protein-like 2 pseudogene; heat shock 27kDa protein 1
SH3 and cysteine rich domain 2
family with sequence similarity 162, member A
cytochrome P450, family 4, subfamily V, polypeptide 2
exocyst complex component 3-like 2
phosphatidylinositol-4-phosphate 5-kinase, type I, gamma
calcium/calmodulin-dependent protein kinase ID
neuronal PAS domain protein 3
activity-regulated cytoskeleton-associated protein
late cornified envelope 1C
activating transcription factor 6 beta
choline dehydrogenase
FtsJ homolog 3 (E. coli)
zinc finger protein 385A
dedicator of cytokinesis 7
calcium regulated heat stable protein 1, 24kDa
PHD finger protein 20
melanoma antigen family E, 1
sorting nexin 10
SH3-domain kinase binding protein 1
chromosome 16 open reading frame 81
family with sequence similarity 129, member B
poly(A) binding protein, cytoplasmic 4-like
malonyl CoA:ACP acyltransferase (mitochondrial)
tripartite motif-containing 44
Cas-Br-M (murine) ecotropic retroviral transforming sequence-like 1
potassium voltage-gated channel, Shaw-related subfamily, member 1
chromosome 21 open reading frame 58
reticulon 3
cleavage stimulation factor, 3' pre-RNA, subunit 2, 64kDa, tau variant
zinc finger protein 124
CD99 molecule
transmembrane and tetratricopeptide repeat containing 2
translocated promoter region (to activated MET oncogene)
transmembrane protein 99
cleavage and polyadenylation specific factor 1, 160kDa
solute carrier family 38, member 7
transmembrane protein 101
testis derived transcript (3 LIM domains)
IQ motif and Sec7 domain 2
adducin 1 (alpha)
adenylate kinase 3-like 2; adenylate kinase 3-like 1
actin, gamma 2, smooth muscle, enteric
taste receptor, type 1, member 1
golgi autoantigen, golgin subfamily a, 8D; golgi autoantigen, golgin subfamily a, 8C; similar to Golgin subfamily A member 8-like protein 1;
golgi autoantigen, golgin subfamily a, 8F; golgi autoantigen, golgin subfamily a, 8G
similar to hCG1993567
lamin B1
exocyst complex component 5
RAB5C, member RAS oncogene family
tetraspanin 13
CD151 molecule (Raph blood group)
epoxide hydrolase 3
metallothionein 1B
hypothetical LOC728675; chromosome 11 open reading frame 75
Rap guanine nucleotide exchange factor (GEF) 1
anterior pharynx defective 1 homolog A (C. elegans)
SRY (sex determining region Y)-box 2
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, assembly factor 4; similar to HSPC125
hypothetical LOC728855
Rho guanine exchange factor (GEF) 16
Supplementary table S.1
metastasis associated lung adenocarcinoma transcript 1 (non-protein coding)
Gene
Symbol
MALAT1
eukaryotic translation elongation factor 1 delta (guanine nucleotide exchange protein)
EEF1D
(De et al., 2006)
apolipoprotein E
APOE
(Sakashita et al., 2008)
transmembrane 4 L six family member 1
TM4SF1
(Kao et al., 2003)
insulin-like growth factor binding protein 5
IGFBP5
(Yasuoka et al., 2009)
fibroblast activation protein, alpha
FAP
(Santos et al., 2009)
AHNAK nucleoprotein
AHNAK
(Shankar et al., 2010)
moesin
MSN
(Estecha et al., 2009)
ectonucleotide pyrophosphatase/phosphodiesterase 2
ENPP2
(Harper et al., 2010)
ets variant 5
ETV5
(Wu et al., 2006)
insulin receptor substrate 2
IRS2
(de Blaquiere et al., 2009)
collagen triple helix repeat containing 1
CTHRC1
(Tang et al., 2006)
matrix metallopeptidase 15 (membrane-inserted)
MMP15
(Mohammad et al., 2010)
matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase)
MMP9
(Liu et al., 2010)
caspase 2, apoptosis-related cysteine peptidase
CASP2
(Guha et al., 2010)
patched homolog 1 (Drosophila)
PTCH1
(You et al., 2010)
SP100 nuclear antigen
SP100
(Negorev et al., 2010)
BCL2-antagonist/killer 1
BAK1
(Zhou et al., 2010)
KISS1 receptor
KISS1R
(Beck and Welch, 2010)
retinol binding protein 1, cellular
RBP1
(Farias et al., 2005)
neuralized homolog (Drosophila)
NEURL
(Teider et al., 2010)
harakiri, BCL2 interacting protein (contains only BH3 domain)
HRK
(Nakamura et al., 2008)
peroxisomal membrane protein 4, 24kDa
PXMP4
(Wu and Ho, 2004)
endothelin 1
EDN1
(Takai et al., 2001)
fragile histidine triad gene
FHIT
(Wali, 2010)
sprouty homolog 2 (Drosophila)
SPRY2
(Holgren et al., 2010)
ADAM metallopeptidase domain 15
ADAM15
(Ungerer et al., 2010)
BCL2 binding component 3
BBC3
(Karst et al., 2005)
HNF1 homeobox A
HNF1A
(Pelletier et al., 2010)
transcription factor AP-2 beta (activating enhancer binding protein 2 beta)
TFAP2B
(Gee et al., 1999)
transgelin
TAGLN
(Prasad et al., 2010)
testis derived transcript (3 LIM domains)
TES
(Gunduz et al., 2009)
spleen tyrosine kinase
SYK
(Layton et al., 2009)
cellular retinoic acid binding protein 2
CRABP2
(Okuducu et al., 2005)
interleukin 27
IL27
(Shimizu et al., 2006)
decorin
DCN
(Bi et al., 2008)
inhibin, beta A
INHBA
(Hong et al., 2010)
adenomatosis polyposis coli 2
APC2
(Senda et al., 2005)
tetraspanin 13
TSPAN13
(Huang et al., 2007)
bone morphogenetic protein 6
BMP6
(Yuen et al., 2008)
kallikrein-related peptidase 3
KLK3
(Mattsson et al., 2009)
Gene Name
References
(Guo et al., 2010)
62