Cell. Mol. Life Sci.

(2013) 70:2677–2696
DOI 10.1007/s00018-012-1187-y Cellular and Molecular Life Sciences
REVIEW

Pleiotropic function of SRY-related HMG box transcription factor

4 in regulation of tumorigenesis
Seyed Mehdi Jafarnejad •
Gholamreza Safaee Ardekani •

Mazyar Ghaffari • Gang Li

Received: 10 June 2012 / Revised: 10 September 2012 / Accepted: 2 October 2012 / Published online: 19 October 2012
Ó Springer Basel 2012

Abstract In addition to their critical roles in embryonic Introduction

development, cell fate decision, and differentiation, mem-
bers of Sox (Sry-related high-mobility group box) family of Sox family of transcription factors
transcription factors including Sox4 have been implicated
in various cancers. Multiple studies have revealed an The Sox (SRY-related HMG box) gene family is found
increased expression along with specific oncogenic func- throughout the animal kingdom. In humans, at least 20
tion of Sox4 in tumors, while others observed a reduced members of this family have so far been identified [1],
expression of Sox4 in different types of malignancies and showing a diverse and dynamic pattern of expression
suppression of tumor initiation or progression by this throughout embryogenesis and in a variety of adult tissue
protein. More interestingly, the prognostic value of Sox4 is types [2]. Sry (for sex-determining region Y), the founding
debated due to obvious differences between various reports member of this family, was identified through searches for
as well as inconsistencies within specific studies. This conserved sequences among translocated Y chromosomal
review summarizes our current understanding of Sox4 DNA from XX male patients [3], and was later confirmed
expression pattern and its transcription-dependent, as well to be involved in male sex differentiation [4].
as transcription-independent, functions in tumor initiation All Sox proteins are characterized by possession of a
or progression and its correlation with patient survival. We high mobility group (HMG) DNA-binding domain. The
also discuss the existing discrepancies between different 79-amino-acid HMG domains bind to the consensus target
reports and their possible explanations. sequence (A/T)ACAA(T/A) in the minor grooves of DNA
[2] and modify the chromatin structure to generate a
Keywords Cancer  Sox4  Oncogene  Tumor suppressor conformation that facilitates various DNA-dependent
activities. This domain is shared with other DNA binding
proteins, including those that bind DNA without sequence
specificity, such as HMG-1 protein and ubiquitous binding
factor (UBF), as well as several sequence-specific DNA-
binding proteins, such as the T cell-specific factors TCF/
LEF [5]. The DNA-binding domains of Sox proteins are at
least 60 % similar or 50 % identical to the HMG box
domain of SRY [6]. The nomenclature of this family is
S. M. Jafarnejad  G. S. Ardekani  G. Li (&)
based on the order of gene discovery [7]. Nine of the 20
Department of Dermatology and Skin Science,
Jack Bell Research Centre, University of British Columbia, human Sox genes contain a single exon, likely reflecting
2660 Oak Street, Vancouver, BC V6H 3Z6, Canada the mechanism of expansion of this ancient gene family via
e-mail: [email protected] non-tandem duplication and retroposition [7]. This family
is sub-grouped into six distinct classes (A–F), based on
M. Ghaffari
The Vancouver Prostate Centre, Vancouver General Hospital, homology within the HMG domain and other structural
University of British Columbia, Vancouver, BC, Canada motifs as well as functional properties. These classes

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2678 S. M. Jafarnejad et al.

include: A, Sry; B, Sox1, -2, -3, -14, -15, and -19; C, Sox4, which are stabilized by a highly conserved cluster of
-11, -12, and -20; D, Sox5, -6, and -13; E, Sox8, -9, and mainly aromatic residues. These hydrophobic cores are
-10; F, Sox7, -17, and -18 [6]. mostly conserved within the HMG box family. Helices I
Despite this common feature, each Sox protein selec- and II are positioned in an anti-parallel mode and form one
tively interacts with and regulates a unique set of target arm of the HMG box, while helix III, which is less rigid,
genes [8]. It appears that Sox proteins are able to bind to a forms an average angle of 90° with the other two helices
large number of transcription factors [9] and on many and constitutes the other arm of the molecule [18]. The
occasions cooperate with them to exert their regulatory N-terminus of the HMG box interacts with the first 6 base
function. For instance, Sox6 was shown to suppress cyclin- pairs of the target sequence, and binding of the HMG box
D1 promoter activity by interacting with b-catenin and to the minor groove of a straight DNA helix in this manner
HDAC1 in pancreatic b-cells [10]. Recruitment of HDAC1 introduces a sharp bend (on the order of 90°) in the DNA
to the promoter regions of the target genes such as DCT helix and alters the local chromatin conformation [18],
and MITF by Sox5 and suppression of their expression was which is crucial for its transcriptional activity.
also observed in melanocytic cells [11]. On the other hand, Another distinctive feature of the SoxC class is a conserved
Sox10 physically interacts with MEF2C and cooperatively proline, serine, and acidic residues-rich transactivation
activates the promoter of the Mef2c gene [12]. domain (TAD) at the C-terminal [16]. Protein secondary
Since the discovery of SRY, many other Sox family structure modeling predicted that 20 residues in the Sox4
members have also been implicated in the regulation of C-terminal domain form an a-helix which is interrupted in the
critical functions in various developmental processes, such middle of its sequence by three randomly coiled residues, and
as sex differentiation, neurogenesis, skeletogenesis, have the last two residues in extended conformation rather
hematopoiesis, angiogenesis, cardiogenesis, melanogene- than in helical conformation [16]. This domain is crucial and
sis, and hair development. Interested readers are referred to sufficient for the activity of SoxC proteins and its deletion
recent reviews on this topic [13–15]. completely abrogates their transactivation capacity [16]. The
Sox4 TAD shows stronger transactivating capacity than
Sox4 Sox12 but weaker than that of Sox11 [16, 17] due to the more
stable a-helix structure of Sox11 TAD [16]. The transacti-
The human Sox4 gene was first identified based on homol- vating function of Sox4 TAD is independent of its HMG
ogy with its mouse homologue [5]. The location of Sox4 was domain which is responsible for DNA binding, as evident
determined by metaphase fluorescence in situ hybridization from its sustained activity upon grafting onto a GAL4 DNA-
(FISH) at chromosome 6p.23 [5]. Sox4 contains a single binding domain [19]. SoxC proteins seem to compete with
exon and its open reading frame (ORF) encodes a protein of each other to bind to their target sequences, since the activity
474 amino acids with a molecular weight of 47 kD [6]. The of any full-length SoxC protein is inhibited by expressing
encoded protein is particularly rich in serine residues (18 % equivalent amounts of Sox4, -11 or -12 proteins lacking the
overall) with several poly-serine stretches. It also includes TAD [16]. The exact mechanism by which TAD regulates
multiple stretches of glycine and alanine residues [5]. transcriptional activation of target genes is not fully under-
Sox4 belongs to the class C (SoxC), which contains two stood. One possible mechanism is that it is achieved through
other members; Sox11 and Sox12 [1]. In 2- to 3-day-old direct interaction with other transcription factors such as p53
mice, SoxC genes are expressed at high levels in the brain. [20] and b-catenin/TCF4 complex [21]. In melanoma cells,
Sox4 and Sox12 are also expressed at a high level in the Sox4 TAD also interacts with syntenin resulting in prevention
heart and lung. While Sox11 is expressed in developing of proteasomal degradation of Sox4, therefore enhancing
limbs, face, and kidneys of the mouse embryo, it is only Sox4 stability [22].
detectable in considerable amounts in neuronal tissues [16] In addition to HMG (aa 57–136) and TAD (aa 441–474)
and is not expressed in detectable amounts in tissues of domains, Sox4 also contains a glycine-rich region (aa
adult mouse, perhaps due to a general decrease in Sox11 152–227) at the center and a serine-rich region (SRR, aa
expression during late embryogenesis [17]. 333–397) adjacent to the TAD (Fig. 1). A study of
All SoxC proteins have a high degree of similarity in the HEK293 cells revealed that the central domain containing a
HMG domain. The HMG box is 84 % identical and 95 % glycine rich region has pro-apoptotic activity which is
similar among SoxC proteins across all vertebrates, independent of the transcriptional activity of Sox4 [23].
whereas their TADs share 67 % identity and 94 % simi-
larity [16]. The structure of the sequence-specific HMG Sox4 in development; jack of many trades
domain of Sox4 provided some insight into its mode of
function [18]. This domain has an L-shaped structure Similar to many other members of the family, the SoxC
consisting of three a-helices connected by loop regions, class has also been identified as a necessary factor in

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Role of Sox4 in tumorigenesis 2679

Fig. 1 Structure of the human Tbx3, Syntenin,

Sox4 protein, its binding Interacting proteins
TCF4, UBC9 GATA-3,
partners, mutations and p53, GATA-3 p53, Brn2
truncated isoform (S395X)
detected in human lung cancer. Sox4 HMG GRR SRR TAD
GRR glycine-rich region, SRR 1 57 136 152 227 333 397 441 474
serine-rich region, TAD A204del A316V
transactivation domain

S395X HMG GRR SRR

1 395

embryonic development. In developing mice, Sox4, -11, Sox4 is expressed in the thymus, bone marrow, and
and -12 are co-expressed at high levels in neuronal and gonads of adult mice [29], which highlights its possible
mesenchymal tissues [16]. The absence of overt phenotype function(s) in hematopoiesis. Early studies showed that
in Sox12-/- mice suggests that at least some functions of Sox4 is highly expressed in thymocytes and induces their
Sox4, -11, and -12 are redundant and Sox4 and -11 may differentiation while promoting pro-B lymphocyte expan-
compensate the loss of Sox12 during mouse development sion [29, 30]. Study of hemopoiesis in lethally irradiated
[24]. Consistently, Bhattaram and colleagues showed that mice reconstituted with Sox4-/- fetal liver cells demon-
concomitant loss of SoxC genes confers a worsened phe- strated that the absence of Sox4 specifically blocks
notype in mouse embryo than loss of each single gene [25]. development of B cells at the pro-B cell stage [26]. Con-
Indeed, the more SoxC genes that are deleted, the more sistently, Sox4-null fetal liver progenitors give rise to all
severe and widespread is organ hypoplasia [25]. SoxC hemopoietic lineages except the B-cell [29]. The pro-B
proteins control neural and mesenchymal cell survival as cells in these mice are moderately reduced in number, but
shown by increased cell death in the neural tube, branchial their capacity to proliferate in response to IL-7 is strongly
arches, and somites of the Sox4-/-11-/- embryos. The abrogated [29]. Sox4 regulates expression of the k5 and
pro-survival role of SoxC proteins in these tissues is VpreB1 genes which form part of the surrogate light chain
probably through direct activation of Tead2, a transcrip- and are expressed in pro- and pre-B cells. k5 and VpreB1
tional mediator of the Hippo signalling pathway which can expression is initially regulated by Sox2 in the pluripotent
promote cell survival during organogenesis [25]. However, cells of the inner cell mass (ICM) of the developing mouse
not all functions of the SoxC proteins seem to be redun- blastocyst, which is then replaced by Sox4 as the cells
dant. Accordingly, Sox4 knockout mice die halfway differentiate toward a very early progenitor for the hema-
through gestation (E14) due to severe heart defect as a topoietic and endothelial lineages, the hemangioblasts [31].
result of impaired development of the endocardial ridges The absence of Sox4 expression may also have some
into the semilunar valves and the outlet portion of the subtle effects on thymocyte development. In fact, thymi
muscular ventricular septum [26] which indicates that from Sox4-null embryos contain two- to four-fold fewer
Sox11 or -12 cannot compensate for the lack of Sox4 cells than thymi from their wild-type or heterozygous
expression in this tissue. The critical role of Sox4 in counterparts [29]. Furthermore, maturation in fetal thymic
development of cardiac tissues may at least partially be due organ culture of mutant thymi is impaired, indicating that
to its role in regulation of expression of the gap junction absence of Sox4 blocks development of T cells from their
protein, connexin 43 (Cx43), in cooperation with the Tbx3 progenitors [29]. A recent study by Kuwahara and col-
transcription factor [27]. leagues [32] revealed that Sox4 inhibits the function of
Sox11-/- mice die at birth from ventricular septation GATA-3, the master regulator of T helper type 2 (Th2)
defects and outflow tract malformations resulting in heart cells, therefore suppressing their differentiation and Th2
defects, in addition to malformation in other organs such as cell-mediated inflammation. In these cells, Sox4 acts as a
skeleton, lung, stomach, and pancreas [28]. The fact that downstream factor of TGF-b and upon activation directly
Sox4 knockout embryos die at E14 and the Sox11-/- mice binds to GATA-3 and prevents its binding to GATA-3
complete the fetal stage indicates that Sox4 has more consensus DNA sequences. In addition, Sox4 binds to the
important roles in early and Sox11 in late embryogenesis. promoter region of the interleukin 5 (IL-5) gene and pre-
Interestingly, SoxC-null embryos will arrest in develop- vents binding of GATA-3 to this promoter, thereby
ment at E8.5 [25], earlier than the embryos lacking each suppressing its expression [32]. It had been previously
individual SoxC member, further confirming that these observed that, during hematopoiesis, the IL-5 receptor
genes have partial overlap in their function which results in activates Sox4 through syntenin-1 [33], perhaps by sup-
exaggerated phenotype upon further loss of each gene. pressing proteasomal degradation of Sox4 protein [22]. It is

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not clear whether these observations, which were made in was described in the musculoskeletal system. Indeed,
different cell types, could indicate the possible existence of 3-month-old Sox4?/- mice have a 64 % lower bone formation
a negative feedback loop between Sox4, IL-5, and IL-5R or rate compared to wild-type, probably due to significant defects
could simply represent different modes of interaction in osteoblast proliferation, differentiation, and mineralization,
between these factors in different cell types. which eventually lead to lower bone mass and to reduced
Sox4 is important for the development of the central trabecular and cortical thickness and growth plate in these
nervous system. In fact, expression of Sox4 and Sox11 is mice [43].
critical for the establishment of pan-neuronal protein The SoxC proteins, including Sox4, are also required for
expression [34]. These proteins act as transcriptional acti- the survival of the multipotent neural and mesenchymal
vators downstream of proneural basic helix-loop-helix stem cells. These cells give rise to various differentiated
(bHLH) proteins to exert this function. Sox4 and Sox11 cell types and are crucial for organogenesis and embryo
exhibit highly overlapping expression in the mouse hip- growth [25]. Importantly, Sox4 is required for maintenance
pocampal neurogenic lineage, and overexpression of either of the stemness of the glioma initiating cells (GICs)
suffices to induce neuronal marker expression in adult through integration of the Oct4/Sox2 axis [44]. The Oct4/
neural stem cells. Consistently, loss of Sox4/Sox11 Sox2 axis is crucial for the continuous self-renewal and
expression results in loss of expression of neuron-specific developmental potential of normal stem cells, specially the
protein DCX, a microtubule-associated protein [35]. embryonic stem cells [45]. However, it is not clear whether
Sox4 and Sox11 are also critical for specification of cor- the Sox4-mediated regulation of stemness is a general
ticospinal neurons partially due to the important role of phenomenon or is limited to the GICs and multipotent
their downstream transcription factor Fezf2 in this process neural and mesenchymal cells.
[36]. Interestingly, this function of Sox4 and Sox11 is
mediated by direct competition with Sox5 which acts as a Regulation of Sox4 expression
repressor of Fezf2 expression [36]. It is also suggested that
Sox4 expression in oligodendrocyte precursor cells may Regulation of Sox4 expression, especially in the patho-
induce premature differentiation of these cells. In fact, logical contexts, is a field that has remained largely
prolonged expression of Sox4 in vivo reduces myelin gene unknown and requires further investigation. Initially,
expression in oligodendrocyte linage which consequently Clarke and colleagues [46] reported that Sox4 is a pro-
interferes with normal myelination in the central nervous gesterone-regulated gene in breast cancer cells and that its
system [37]. Interestingly, expression of Sox4 and Sox11 in expression is induced by progestins. Consistently, treat-
neural progenitor cells is restricted by REST/NRSF [34]. ment of T-47D breast cancer cells with the synthetic
REST/NRSF is a transcriptional repressor which restrains progestin ORG-2058 increased Sox4 transcription within
the neurogenic program in progenitor cells [38], and few hours of treatment. Notably, ORG-2058 had no
repression of Sox4 and Sox11 by this protein may con- detectable effect on the expression of other Sox genes,
tribute to the mechanism by which REST/NRSF prevents suggesting that in this system the observed phenomenon
expression of neuronal proteins in these cells. was specific to Sox4 [46]. In osteoblast-like cells, physio-
Other than the nervous system, Sox4 has a number of logical concentrations of human parathyroid hormone
important roles in other developmental processes. During stimulate Sox4 mRNA expression in a time-dependent
mouse development, Sox4 expression is more confined to manner, indicating involvement of the PTH/PTHrP recep-
the pancreatic epithelium and later islet cells along with tor [47]. Prostaglandin A2 and delta12-PGJ2 also induce
Sox9 expression [39]. Sox4b (an isoform of the zebrafish Sox4 mRNA expression in hepatocarcinoma cells [48]. Del
homologue of Sox4) is also a key player in pancreatic alpha Giacco and colleagues found that IFN-beta/all-trans reti-
cell differentiation [40]. In adult mice, Sox4 is broadly noic acid treatment induced the expression of Sox4 in a
expressed in the early pancreatic buds and in the nuclei of thioredoxin reductase 1 (TrxR1) enzyme-dependent man-
all islet cells [41]. Homozygous deletion of Sox4 did not ner in hepatocarcinoma cells [49]. However, in none of
show any abnormalities in pancreas development up to these observations is it clear whether these hormones affect
embryonic day 12.5; however, a significantly reduced the activity of the Sox4 promoter or whether they exert
number of endocrine cells were found to be scattered their function at other levels (e.g., mRNA stability).
through the culture site, causing failure to form normal Nonetheless, other studies have revealed details on the
islets [41]. This phenomenon significantly affects normal regulation of the Sox4 promoter. In hematopoietic pro-
pancreatic function, as it has been proven that Sox4 dele- genitor cells, Sox4 expression is regulated at transcription
tion in adult mouse results in a considerable defect in level by the HOXB4 transcription factor [50]. The HOX
insulin secretion and leads to impaired glucose tolerance [42]. gene family members are important regulators of hemato-
The other important role of Sox4 in the developmental process poiesis. In T cells, expression of Sox4 is upregulated at

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Role of Sox4 in tumorigenesis 2681

both mRNA and protein levels by TGF-b. This regulation before the emergence of evidence that the developmentally
is mediated by downstream mediators of TGF-b, Smad2 critical Sox proteins may also play a role in some cancers.
and Smad3, which bind to the regulatory region of the Sox4 Several lines of evidence pointed to the fact that Sox
gene [32]. Overexpression of the Sox4 transcript upon proteins may be implicated in cancer. For instance,
binding of the Smad2/3 complex to its promoter after TGF- amplification of Sox2 at chromosome 3q has been detected
b treatment has also been observed in glioma cell lines in prostate cancer [61]. The chromosomal region 20q13,
[51]. On the other hand, inhibitors of differentiation 2 which contains Sox18 coding gene, is also amplified in
(ID2) and REST/NRSF repress expression of Sox4 in some cases of breast [62] and colon cancers [63]. Dereg-
neural progenitor cells [34]. Since REST/NRSF is a tran- ulated expression of other members of the Sox family in
scriptional repressor, it is pertinent to assume that it could malignancies has also been demonstrated [64–66]. Keeping
suppress Sox4 promoter activity, although this assumption in mind that in many cases the expression status of Sox
requires further experimental verification. The retinoic acid genes differs in various cancers, it would be implausible to
receptor-related orphan receptor a (RORa) is also sug- assume a ubiquitous oncogenic or tumor suppressive
gested to upregulate Sox4 transcription [52]. RORa plays a function for any given Sox gene. This kind of inconsistency
critical role in regulation of the circadian clock and in the expression pattern and mode of function (oncogenic
enhances Sox4 transcription by direct binding to the pro- vs. tumor suppressive) in different types of cancer exists
moter sequences after activation by its agonist [52]. for several members of the Sox family, especially Sox4,
Sox4 is transcriptionally regulated by Sox7 in endome- which will be described below in more detail.
trial cancer cells [53]. Accordingly, overexpression of Sox7
activates the Sox4 promoter, leading to the increased Oncogenic functions of Sox4
expression at both mRNA and protein levels, seemingly
due to the presence of Sox7-responsive elements in the Several lines of evidence suggest an oncogenic role for
promoter region of Sox4 gene. Interestingly, Sox7 inhibits Sox4 in a wide range of cancers. Some of the earliest
the activity of its own promoter [53], further confirming indirect evidences highlighting a role for Sox4 in cancer
that the same Sox protein can simultaneously enhance and came from studies on colon carcinoma by Miyamoto and
suppress the activity of different promoters in the same colleagues [67]. In this study, Sox4 was found to enhance
cell, consistent with the context-dependent manner of transcription of p56lck, a member of the family of Src
function for Sox proteins. Sox4 expression may further be tyrosine kinases that is aberrantly expressed in colon and
influenced by other members of the SoxC class. In fact, small lung carcinoma cell lines. p56lck is also expressed in
whereas ablation of Sox4 in the sympathoadrenal lineage T cells and is among the first signaling molecules to be
did not have any significant effect on expression of Sox11, activated downstream of the T cell receptor and plays a
knockdown of Sox11 markedly reduced Sox4 protein significant role in T cell differentiation, survival, and
expression [54], although it is not clear whether or not this activation [68]. In Jurkat and HeLa cells, expression of
effect of Sox11 is exerted through direct binding to the p56lck is controlled by Myb, in synergy with Ets factors
Sox4 promoter. In addition, Sox4 may regulate its own [69]. However, in colon carcinoma, aberrant expression of
expression as is evident by binding of the Sox4 protein to p56lck arises from transcriptional activation mediated by
its promoter sequence in prostate cancer cells [55]. cooperation between Ets-1 and Sox4 [67]. Amplification of
Other mechanisms also contribute to the regulation of chromosomal region 6p22.3 in at least some tumor types
Sox4 expression at post-transcriptional and post-transla- [70, 71] was another indirect evidence for a tumorigenic
tional levels, which are summarized in Fig. 2 and will be function of Sox4. Nevertheless, in some cases, there is no
discussed in detail throughout this review. significant correlation between expression of Sox4 and
amplification of 6p22.3 [73, 74], suggesting that amplifi-
Role of Sox4 in tumorigenesis cation of Sox4 coding sequences might be a bystander
effect and not a growth advantage for some cancer cells.
In retrospect, many genes that are involved in develop- This suggestion is in line with existence of important proto-
mental processes have also been found to play key roles in oncogenes such as E2F3 [72], ID4 [73] and, PRL [74] in
the formation of malignancies. For instance, the hedgehog the 6p22.3 chromosomal region, which might provide
pathway was originally discovered in Drosophila, in which the actual benefit to the cancer cells upon amplification.
its members encode segment polarity proteins responsible This notion is consistent with an independent observa-
for determining the anterior–posterior orientation of the tion in bladder cancers. In this cancer, 30.8 % of the
Drosophila embryo and larva [56], and was later found to patients had chromosomal amplification of this region. The
be implicated in tumorigenesis [57, 58]. Involvement of the mean E2F3 expression of amplified tumors was a 1.8-fold
HMG protein family in cancer was already known [59, 60] increase compared with not-amplified tumors, whereas no

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2682 S. M. Jafarnejad et al.

DNA damage

Sox4

miR-93, miR-129-2,

Syntenin
Progesterone, PTH,
PGs, IFN- , RA miR-335
Proteasome

HOX4B Smad 2/3 Sox7 ROR

Sox4 transcript
Sox4 Sox4 promoter

Proteasomal REST/NRSF ID2

degradation
Sox4 promoter

Fig. 2 Regulation of Sox4 expression. Expression of Sox4 is regulated at three distinct levels; transcriptional, post-transcriptional, and
post-translational. PGs prostaglandin A2 and delta12-prostaglandin J2

correlation was found between chromosomal amplification role of Sox4 in those cells. For instance, Sox4 protein is
and Sox4 expression [75]. Nonetheless, the inconsistency overexpressed in human hepatocellular carcinomas (HCC)
between 6p amplification and Sox4 expression may not be [77], yet there is an inverse correlation between expression
a ubiquitous phenomenon. In fact, another study by Medina of Sox4 and recurrence of cancer as well as a positive cor-
et al. [76] found an increased expression of Sox4 in lung relation between Sox4 expression and overall patient
primary tumors and lung cancer cell lines with chromo- survival which does not fit into a possible oncogenic role for
some 6p amplification. Interestingly, they also reported Sox4 in this type of cancer. This study also found that the
identification of a somatic mutation at the 395 residue of HMG box domain of Sox4 interacts with p53. This inter-
Sox4 that introduces a premature stop codon at the C-ter- action, which happens at the p53-responsive promoters,
minal domain, producing a shorter Sox4 protein, S395X, results in inhibition of p53-mediated activation of Bax
which is devoid of the region immediately after the serine- promoter, leading to repression of p53-induced Bax
rich domain and does not possess transactivation potential expression and subsequent repression of p53-mediated
(Fig. 1). While neither wild-type Sox4 nor its truncated apoptosis induced by gamma irradiation. Therefore, Sox4
isoform could induce oncogenic transformation of the suppresses p53-mediated apoptosis induced by gamma
NIH3T3 mouse fibroblasts, wild-type Sox4 enhances irradiation in HCC cells [77]. Of note, this function of Sox4
tumorigenicity of the NIH3T3 cells expressing the acti- in HCCs is in sharp contrast with its role in colorectal cancer
vated HRAS. On the other hand, the truncated isoform in which Sox4 interacts with and stabilizes p53 activity and
significantly reduced the tumorigenicity of the HRAS- enhances p53-mediated apoptosis, cell cycle arrest, and
expressing cells [76], which suggests that transcriptional inhibits tumorigenesis [20].
activity of Sox4 supports the HRAS-transforming ability in A tissue microarray analysis of 2,360 samples revealed
lung cancer cells. However, it is not clear why cancer cells an increased expression of Sox4 protein in bladder tumors,
with a truncated Sox4 that could reduce the tumorigenicity suggesting an oncogenic role for Sox4 in this type of tumor
would have a selective advantage. [75]. Nevertheless, this notion is in contrast with survival
Increased expression of Sox4 at mRNA or protein levels studies in the same set of patients where a significant
in several types of epithelial and hematopoietic cancers correlation between strong Sox4 expression and increased
further confirmed its putative oncogenic function (Table 1). patient survival was found, probably due to the role of
However, this observed correlation between Sox4 overex- Sox4 in induction of apoptosis in bladder carcinoma cells.
pression and cancer progression does not seem to always be Indeed, in these cells, Sox4 overexpression impairs cell
consistent with patient survival outcome or even functional viability and enhances cell death [75]. This study also

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Role of Sox4 in tumorigenesis 2683

Table 1 Expression pattern and biological functions of Sox4 in different types of malignancies and its correlation with patient survival
Type of tumor Sox4 Method of detection Suggested function Correlation with Reference
expression survival

Acute myeloid : Western blot Promotion of cell growth ND [96]

leukemia
Adenoid cystic : cDNA microarray ND ND [82]
carcinoma
Adenoid cystic : Tissue microarray Suppression of apoptosis ND [81]
carcinoma
Bladder carcinoma : cDNA microarray/tissue microarray Induction of apoptosis Positive [75]
Colon ; cDNA microarray ND ND [131]
adenocarcinomas
Colorectal cancer : Tissue microarray ND ND [112]
Colorectal cancer : cDNA microarray ND Negative [112]
Cutaneous melanoma ; Tissue microarray Suppression of invasion and Positive [104]
migration
Endometrial cancer : Tissue microarray Promotion of cell growth ND [85]
Hepatocellular : Tissue microarray Suppression of apoptosis Positive [77]
carcinoma
Gallbladder ; IHC ND Positive [110]
carcinoma
Gastric cancer : Tissue microarray Suppression of apoptosis Negative [86]
Glioblastoma : Parallel signature sequencing/ ND ND [99]
RT-PCR/IHC
Lung cancer : RT-PCR/western blot Induce transformation of ND [76]
NIH3T3 cells
Medulloblastoma : RT-PCR/ISH ND ND [65]
Medulloblastoma : RT-PCR ND ND [132]
Pediatric : cDNA microarray ND Positive [111]
medulloblastoma
Prostate cancer : cDNA microarray ND ND [78]
Prostate cancer : Tissue microarray/cDNA Suppression of apoptosis ND [79]
microarray
Uveal melanoma ; Suppressive subtractive ND ND [105]
hybridization
ND not determined, ISH in situ hybridization, IHC immunohistochemistry

found that, in bladder cancer cells, Sox4 regulates a suppresses apoptosis and enhances transformation of
plethora of genes including transcription factors, such as prostate cancer cells. However, the mechanism by which
MEF2C and zinc finger protein 6 (ZNF6), and signaling Sox4 regulates p53 expression in these cells is not under-
molecules such as phosphoinositide-3-kinase regulatory stood. Further analysis by cDNA microarray of LNCaP
subunit polypeptide 3 (PIK3R3), that promotes cell cycle cells treated with Sox4 siRNAs or overexpressing Sox4
arrest, and mitogen-activated protein kinase kinase 5 revealed 466 genes which were consistently responsive
(MAP2K5) [75]. both to Sox4-knockdown and overexpression. Among the
An oncogenic role for Sox4 in prostate cancer and its Sox4 target genes identified in this study were genes with
progression is well established [78, 79]. Moreno and col- roles in Wnt signaling (TLE-1), apoptosis (BCL10 and
leagues reported that Sox4 is the only member of the Sox PUMA), and inflammation (CSF1) [79].
family that is overexpressed in prostate cancer, and its The same group later performed a genome-wide pro-
knockdown induces apoptosis in the LNCaP prostate can- moter analysis of the Sox4 transcriptional network in
cer cell line. Consistently, Sox4 overexpression induced prostate cancer cells and identified 3,470 different genes
transformation of RWPE-1 prostate cancer cells. Further- whose promoter sequences contain direct binding sites of
more, depletion of Sox4 expression resulted in an increased Sox4. However, cDNA microarray analysis revealed that,
expression of p53 protein and loss of survivin expression among these genes, expression of only 282 of the 3,470
[79], possibly explaining the mechanism by which Sox4 binding targets is significantly influenced by Sox4 [80].

123
2684 S. M. Jafarnejad et al.

This observation suggests that a change in expression of (LNCap) only returned three common genes (Fig. 3).
Sox4 is not sufficient to alter the expression status of the Bearing in mind that these comparisons should be con-
other 3,188 genes, reflecting the requirement for other sidered carefully due to differences in the technical aspects,
regulatory components such as binding partners in this such as the type of microarray platforms or the time points
process. Among the genes which were shown to be directly after overexpression or knockdown of Sox4 at which the
regulated by Sox4 in this study are imminent proto-onco- RNA samples were collected, these results are also another
genes such as Tenascin-C, Frizzled-3, 5 and 8, Patched-1, indication of the highly tissue- and cell-specific network of
Delta-like1, and EGFR, which are positively regulated by Sox4.
Sox4 and reduced after Sox4-knockdown in prostate cancer As mentioned earlier, expression of Sox4 is regulated at
cells [80]. The extent of the Sox4 network encompasses post-transcriptional level. For instance, miR-129, which is
various cancer-related pathways and mechanisms including overexpressed in bladder carcinoma and inversely corre-
the TGF-b, Wnt, hedgehog, and Notch pathways, as well as lated with patient survival, targets Sox4 in these cells [84].
growth factor signaling and tumor metastasis. Notably, this Similarly, expression of miR-129-2 is lost in the majority
study revealed differential regulation of expression of tar- of primary endometrial tumors, probably due to hyperme-
get genes by Sox4 in different prostate cancer cell lines. thylation of the miR-129-2 regulatory regions, with a
For instance, Sox4 binds to the promoter sequences of concomitant gain of Sox4 expression [85]. Restoration of
EGFR and enhances its expression in RWPE-1 cells, yet it miR-129-2 expression in these cells leads to decreased
does not bind to these sequences in LNCaP cells [80]. Sox4 expression and reduced proliferation of cancer cells.
Interestingly, Sox4 overexpression increases expression of Consistently, Sox4 protein is overexpressed in endometrial
the transcription factor SON by 1.8-fold, whereas its tumors [85], and its knockdown suppresses endometrial
knockdown increases expression of purine biosynthetic cancer cell growth. Downregulation of Sox4 by miR-129-2
enzyme GART by threefold. These two genes, located on as well as hypermethylation-mediated suppression of this
chromosome 21, are regulated by a common bidirectional miRNA is also described in gastric cancers, in which both
promoter but transcribed in opposite directions. Differen- Sox4-knockdown and overexpression of miR-129-2 induce
tial regulation of expression of these two genes by Sox4 apoptosis [86].
suggests that it regulates the directionality of this promoter In a search for miRNAs which regulate cancer growth
[80]. This study also identified co-occurring binding sites and metastasis, Tavazoie et al. [87] found that expression
of several transcription factors such as E2F1, E2F4, PAX5, of miR-335 is lost in the majority of primary breast tumors,
LEF1/TCF1, and MYC along with Sox4 in its target pro- and that this loss of expression is associated with poor
moter [80], which highlights the possibility of cooperation distal metastasis-free survival. In this type of cancer, miR-
between Sox4 and one or more of these factors in regula- 335 regulates a set of genes, including Sox4 and the
tion of the expression of target genes. extracellular matrix component Tenascin C, whose
Sox4-knockdown causes a considerable reduction in expression in a large cohort of human breast tumors is
adenoid cystic carcinoma (ACC) cell viability due to associated with risk of distal metastasis. Consistently,
induction of apoptosis in a caspase-dependent manner [81]. knockdown of Sox4 or Tenascin C significantly abolishes
In concert with this observation, both Sox4 transcript and lung colonization by LM2 breast cancer cells. Interestingly,
protein were found to be upregulated in ACC [81, 82]. in the genome-wide promoter analysis in prostate carci-
Microarray gene expression profiling of Sox4-knockdown noma cells, Sox4 was shown to bind to the promoter
ACC cells confirmed Sox4-modulated expression of criti- sequences of Tenascin C [80]. The study by Tavazoie and
cal genes involved in apoptosis and cell cycle control such colleagues showed that miR-335 directly targets Sox4.
as cyclin G2 (CCNG2), DUSP4, and BNIP3, indicating the However, it is not clear whether downregulation of
contribution of Sox4 to the malignant phenotype of ACC Tenascin C by miR-335 is a direct event or is achieved
cells by promoting cell survival. This study, along with through initial downregulation of Sox4, resulting in reduced
other cDNA microarray analyses in human prostate cancer expression of Tenascin C.
[79, 80], bladder cancer [75], and small cell lung cancer In addition to miR-335, in breast cancer cells miR-93
[83] cells, identified a large number of genes, the expres- also targets Sox4. This effect may be important for the
sion of which is affected by Sox4. Although each study observed induction of mesenchymal–epithelial transition
identified a considerable number of target genes, the (MET) associated with downregulation of TGF-b signaling
overlaps between these datasets are almost negligible in breast cancer cells and resulting in cancer stem cell
(Fig. 3). In fact, there is no gene whose expression is at depletion [88]. In line with this observation, Sox4 may
least two-fold changed upon overexpression or knockdown contribute to invasion and metastasis of breast cancer cells
of Sox4 in all five datasets. Even more surprisingly, com- by induction of epithelial-mesenchymal transition (EMT).
parison of two studies in the same prostate cancer cell line In fact, overexpression of Sox4 in immortalized human

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Role of Sox4 in tumorigenesis 2685

By a sophisticated approach, using oncogenic retrovirus-

induced insertional mutagenesis, Copeland and colleagues
[91] showed that replication-defective retrovirus carrying
Sox4 can induce myeloid leukemias after transplanting into
mice bone marrow cells in cooperation with Mef2c, a
transcription factor involved in regulation of homing and
invasiveness of leukemic cells. Sox4 can integrate at the
site of Sfpi1, an Ets1-related transcription factor essential
for normal myeloid and lymphoid development, and reduce
Sfpi1 expression [92]. The same group later revealed that
Sox4 represses Sfpi1 transcription by binding to a critical
Sfpi1 upstream DNA element [93]. Additionally, by ana-
lyzing 285 acute myeloid leukemia (AML) patient samples,
they found a significant negative correlation between Sox4
and Sfpi1 mRNA expression providing convincing evi-
dence for the involvement of Sox4 in promotion of at least
a subset of myeloid malignancies [93]. Sox4 is the site of
the Evi1 (ectopic viral integration site 1) proviral insertions
in myeloid leukemias. This insertion results in transcrip-
tional activation and increased Sox4 expression [91].
Interestingly, Sox4 expressing myeloid cells have a higher
level of transcripts associated with proliferation including
Evi1, despite no measurable effect on cell proliferation or
differentiation after overexpression of Sox4 [91].
Sox4 is also reported to be a common retrovirus inser-
tion site in myeloid and monocytic cells [94], suggesting a
role of Sox4 in promoting malignant disease in these cells.
Consistently, retroviral tagging strategy in combination
with high throughput inverse-PCR identified Sox4 as one
of the most common retroviral integration sites in a tumor
panel composed primarily of B cell lymphomas [95]. In
myeloid cells, Sox4 binds to the promoter sequences of
CREB and enhances its expression [96]. Transduction of
CREB transgenic mouse bone marrow cells with a Sox4
retrovirus increases the in vitro survival and self-renewal.
In addition, leukemic blasts from the majority of AML
Fig. 3 Comparative analysis of cDNA microarray studies of Sox4
target genes in prostate cancer, adenoid cystic carcinoma, bladder,
patients have higher CREB, phosphorylated CREB, and
and small cell lung cancer cell lines. Target genes whose expression Sox4 protein expression, indicating that Sox4 and CREB
was [two-fold changed after Sox4 overexpression [75, 79, 80] or cooperate and contribute to increased proliferation of
knockdown [81, 83] and represented in at least two datasets were hematopoietic progenitor cells and myelogenesis.
included in this analysis
Sox4 also binds to and activates the promoter of CD56
in primary myeloma cells [97]. CD56 is a member of the
mammary epithelial cells is sufficient for acquisition of immunoglobulin superfamily that was initially character-
mesenchymal traits, enhanced cell migration, and inva- ized in the cells of the nervous system and is overexpressed
sion [89]. Moreover, based on tumorigenesis assay in in more than 80 % of myeloma patients as well as some
mice, Sox4 cooperates with activated Ras to promote other types of malignancies [98]. Thus, it would be of
tumorigenesis in vivo [89]. In addition to breast cancer, interest to investigate the possible role of Sox4 in regula-
Sox4 also augments the migration, invasion, and metastasis tion of CD56 expression in other types of cancer.
of hepatocellular carcinoma cells [90]. This function of
Sox4 is probably exerted by upregulation of two down- Promotion of tumorigenesis by sustainment of stemness
stream factors, neuropilin 1 (NRP1) and semaphorin 3C
(SEMA3C), knockdown of which drastically reduces cell In another study delineating the molecular details of Sox4’s
migration [90]. involvement in tumorigenesis, Ikushima et al. [51] found

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2686 S. M. Jafarnejad et al.

that Sox4 is highly expressed in GICs and required for the nude mice as a xenograft from Hep3B cells [48], which is
TGF-b-induced expression of Sox2 that is essential for not consistent with a tumor suppressor role for Sox4.
retention of stemness in GICs. Consistently, Sox4-knock- Unfortunately, the authors did not investigate this contra-
down cells show less sphere-forming ability and self- diction in their results in further detail. The same group
renewal capacity, two characteristic features of GICs. Sox4 later observed that overexpression of Sox4 induces sig-
regulates Sox2 expression at transcriptional level by nificant apoptosis in Hep3B and HepG2 cell lines, probably
binding to an enhancer element located at its 30 flanking through the caspase-mediated pathway [100]. In addition,
region. This activity is increased after TGF-b stimulation. Sox4-knockdown blocks the apoptosis induced by PGA(2)
In addition, Sox4 mRNA expression is immediately and delta(12)-PGJ(2) in these cells [100]. The caspase-
induced after TGF-b stimulation through binding of dependent manner of Sox4 for induction of apoptosis was
Smad2/3, the DNA-binding mediators of TGF-b signaling. later confirmed by Kim et al. [101]. Interestingly, although
Consistently, inhibitors of TGF-b signaling drastically several prominent apoptosis-related factors such as Bax
deprived tumorigenicity of GICs by promoting their dif- and PUMA are among the direct transcriptional targets of
ferentiation, while these effects are attenuated in GICs Sox4 (Table 2), the pro-apoptosis function of Sox4 can be
overexpressing Sox2 or Sox4 [51]. The same group later dissociated from its transcriptional activity [23]. Indeed,
demonstrated that Oct4 physically interacts with Sox4 and the central domain (aa 166–342) of Sox4 is critical for
cooperatively activates the Sox2 enhancer region to induction of apoptosis in HEK293 cells. Accordingly,
maintain stemness properties of GICs [44]. Supporting this deletion of the DNA-binding domain or trans-activation
notion, consensus sequences of Sox proteins and Oct4 exist domain in Sox4 does not significantly affect its pro-apop-
in close proximity of each other in the Sox2 enhancer totic activity in these cells, whereas overexpression of a
region, and ChIP re-IP (back-to-back immune-precipitation construct containing the central domain induces the apop-
of chromatin with two different antibodies) assay demon- totic activity comparable to that of the full-length protein
strated that Sox4 and Oct4 exist in the same transcription [23].
complex in the Sox2 enhancer region and enhance the As an indirect link between Sox4 and regulation of
activity of Sox2 promoter [44], further confirming the role apoptotic cell death, human ubiquitin-conjugating enzyme
of Sox4 in regulation of Sox2 expression and mainte- 9 (Ubc9) interacts with HMG-box of Sox4 and represses
nance of GICs’ tumorigenicity. A separate study identified Sox4 transcriptional activity in HEK293T cells [102]. The
elevated expression of Sox4 and TGF-b in human glio- C93S mutant of Ubc9, which abrogates SUMO-1 conju-
blastoma compared with normal brain tissues at both RNA gation activity, does not abolish its ability to inhibit Sox4
and protein levels, and confirmed that Sox4 expression is activity. Elevated Ubc9 expression has been detected in at
increased by TGF-b stimulation [99]. least some types of malignancies, such as primary and
In breast cancer, Sox4 overexpression induces EMT, metastatic melanomas in which Ubc9 plays a crucial role in
resulting in a cancer stem cell-like phenotype in breast suppression of apoptosis [102]. On this note, in some
cancer cells. These cells exhibited an increase in the CD44 malignancies such as breast cancer [103], the ability of
high/CD24 low sub-population and enhanced size and Ubc9 to promote cancer initiation and progression is
number of mammospheres, indicating that the Sox4- independent of its sumoylation function. The possible
induced EMT generates mesenchymal cells with stem cell- involvement of Sox4 inactivation in the sumoylation-
like phenotype [89]. This function of Sox4 may also be independent oncogenic function of Ubc9 remains to be
mediated by the TGF-b pathway as is evident by increased uncovered.
expression of TGF-b1 and TGF-b2 mRNAs and the pho- Our own studies on cutaneous malignant melanoma have
spo-Smad2 protein in Sox4-expressing cells [89]. revealed that Sox4 expression is significantly reduced in
metastatic melanomas [104]. Furthermore, Sox4 expression
Sox4 as a tumor suppressor is positively correlated with better patient survival. Con-
sistently, Sox4 suppresses melanoma cell migration and
The first evidence to pinpoint a possible role for Sox4 in invasion ability through inhibition of NF-jB p50 expression
suppression of tumorigenesis came from a study by Ahn by binding to sequences upstream of the NF-jB p50 pro-
et al. [48]. Using mRNA differential display and northern moter [104]. A recent study reported a reduced expression
blot analysis, they revealed that expression of Sox4 mRNA of Sox4 mRNA in uveal melanoma [105]. Overexpression
is enhanced during apoptosis induced by prostaglandin of several members of the NF-jB pathway has also
(PG)A2 and Delta12-PGJ2 in human hepatocellular carci- been observed in metastatic uveal melanoma [106]. How-
noma cells, Hep3B, suggesting the possible involvement of ever, it is not clear whether or not Sox4-mediated inhibition
Sox4 in the process of apoptosis. Nevertheless, Sox4 was of NF-jB can play a role in suppression of this type of
also highly expressed in subcutaneous tumors grown in melanoma.

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Role of Sox4 in tumorigenesis 2687

Table 2 Validated direct transcriptional targets of Sox4

Tissue/cell type Gene symbol Mode of regulation Function Assay References

Breast cancer ZEB1 : Transcription repressor Reporter assay [89]

Breast cancer Snail : Transcription repressor Reporter assay [89]
Colon carcinoma p56lck : Protein tyrosine kinase Reporter assay [67]
COS1 Tubb3 : b-tubulin isotype III EMSA [34]
Endometrial carcinomas TCF4 : Transcription factor Reporter assay [53]
Glioma Sox2 : Transcription factor ChIP [51]
HCC SLC2A1 : Agmatine transport ChIP [90]
HCC Bax ; Apoptosis ChIP [77]
HCC CREB3L1 : Axon guidance ChIP [90]
HCC MYEF2 : Axon guidance ChIP [90]
HCC NAV3 : Axon guidance ChIP [90]
HCC NRP1 : Axon guidance ChIP [90]
HCC SEMA3C : Axon guidance ChIP [90]
HCC NEIL3 : Base excision DNA repair ChIP [90]
HCC CHFR : Cell cycle checkpoints ChIP [90]
HCC NPNT : Extracellular matrix protein ChIP [90]
HCC GPRC5B : G-protein signaling pathway ChIP [90]
HCC HUNK : Kinase ChIP [90]
HCC PFKFB4 : Kinase ChIP [90]
HCC AKR1B10 : Metabolism ChIP [90]
HCC ALDH18A1 ; Metabolism ChIP [90]
HCC DHRS13 : Metabolism ChIP [90]
HCC GYG : Metabolism ChIP [90]
HCC PAM : Metabolism ChIP [90]
HCC RCC2 ; Metabolism ChIP [90]
HCC MAP4 : Microtubule-associated protein ChIP [90]
HCC SMG5 : mRNA decay ChIP [90]
HCC HSPBAP1 ; Protein folding ChIP [90]
HCC LCK : Protein tyrosine kinase ChIP [90]
HCC CSPG2 : Proteoglycan ChIP [90]
HCC CTSC : Proteolysis ChIP [90]
HCC RBM10 ; RNA binding ChIP [90]
HCC SERPINE2 : Serine protease inhibitor ChIP [90]
HCC INST8 : Small nuclear RNAs processing ChIP [90]
HCC HIC2 ; Transcription factor ChIP [90]
HCC VGLL4 : Transcription factor ChIP [90]
HCC FOXQ1 : Transcription factor ChIP [90]
HCC TEAD2 : Transcription factor ChIP [90]
HCC CCDC97 ; Unknown ChIP [90]
HCC UBAP2L ; Unknown ChIP [90]
HCC DKK1 : Wnt signaling pathway ChIP [90]
Heart Connexin 43 : Gap junction protein ChIP [27]
Hemangioblasts k5 : Component of precursor of BCR ChIP [31]
Hemangioblasts VpreB1 : Component of precursor of BCR ChIP [31]
Melanoma NF-jB p50 ; Inflamation, cell growth ChIP [104]
Melanoma Dicer : miRNA biogenesis ChIP [107]
Mouse embryonic mesenchymal cells Tead2 : Transcription factor ChIP [25]
Myeloid Leukemia CREB : Transcription factor ChIP [96]

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2688 S. M. Jafarnejad et al.

Table 2 continued
Tissue/cell type Gene symbol Mode of regulation Function Assay References

Myeloid leukemias Sfpi1 ; Transcription factor ChIP [93]

Myeloma CD56 : Immunoglobulin superfamily ChIP [97]
Neural cells DCX : Microtubule-associated protein ChIP [35]
Neural cells GnRH : Sexual development ChIP [133]
Neural cells FEZF2 : Transcription factor ChIP [36]
Prostate PUMA : Apoptosis ChIP [79]
Prostate MLL : Histone methyl-transferase ChIP [80]
Prostate ADAM10 : Metalloproteinase ChIP [80]
Prostate AGO1 : miRNA biogenesis ChIP [80]
Prostate DHX9 : miRNA biogenesis ChIP [55, 80]
Prostate Dicer : miRNA biogenesis ChIP [55, 80]
Prostate DLL1 : Notch signaling ChIP [80]
Prostate HES2 : Notch signaling ChIP [80]
Prostate RBL1 : Regulation of cell cycle ChIP [80]
Prostate EGFR : Signaling ChIP [80]
Prostate ELF5 ; Transcription factor ChIP [80]
Prostate Sox4 : Transcription factor ChIP [55]
Prostate ZNF281 : Transcription repressor ChIP [80]
Prostate TLE-1 : Transcription repressor ChIP [79, 80]
Prostate FZD3 : Wnt receptor ChIP [80]
Prostate FZD5 : Wnt receptor ChIP [80]
Prostate FZD8 : Wnt receptor ChIP [80]
Prostate AXIN2 : Wnt signaling ChIP [55]
SCLC VASH2 : Angiogenic factor ChIP [83]
SCLC PCDHB : Protocadherin ChIP [83]
SCLC Tead2 : Transcription factor ChIP [83]
SCLC MYB : Transcription factor ChIP [83]
SCLC Tubb3 : b-tubulin isotype III ChIP [83]
T-cells Interleukin 5 ; Cytokine ChIP [32]
T-Cells CD2 : Surface antigen EMSA [134]
EMSA electrophoretic mobility shift assay, ChIP chromatin immunoprecipitation, HCC hepatocellular carcinoma

In addition, Sox4 may recruit miRNA machinery to suppressor in response to DNA damage (discussed in fur-
negatively regulate melanoma invasion. In cutaneous ther details below).
melanoma cells, Sox4 regulates transcription of Dicer by Regulation of expression of miRNA-related factors such
binding to its promoter and enhancing its activity which is as Dicer, Argonaute 1, and RNA helicase A by Sox4 had
critical for expression of a considerable number of cancer- been previously reported in prostate cancer cells by Scharer
related miRNAs [107]. Interestingly, Dicer and Drosha et al. [80]. Although there were some inconsistencies in the
have recently been identified to be necessary to activate the pattern of Sox4 target genes between different cell lines,
DNA repair mechanism upon exogenous DNA damage and they were able to confirm the regulation of Dicer expres-
oncogene-induced genotoxic stress [108], although this sion by Sox4 at both transcript and protein levels [80],
function appears to be independent from the canonical which was consistent with our observation in melanoma
miRNA-mediated translational repression mechanisms cells [107]. It is noteworthy that, while we observed that
regulated by Dicer. This mechanism may also be respon- Dicer expression is lost in metastatic melanoma and it is
sible for Sox4-mediated suppression of tumorigenesis, required for Sox4-mediated suppression of melanoma cell
considering the regulation of Dicer expression by Sox4 and invasion, expression of Dicer is upregulated in prostate
the fact that Sox4 has also been reported to be a DNA adenocarcinomas [109]. Although the expression status of
damage sensor and is required for activation of p53 tumor Dicer is not known in other subtypes of prostate cancer,

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Role of Sox4 in tumorigenesis 2689

this observation may be related to overexpression of Sox4 p53 acetylation which increases p53 stability. In this sys-
[79] and highlights the importance of Sox4-regulated Dicer tem, Sox4 promotes cell cycle arrest and apoptosis, and
expression in these two different types of malignancies. inhibits tumorigenesis in a p53-dependent manner [20]. In
In primary gallbladder carcinoma, Sox4 expression is line with these observations, ionizing radiation induces
reduced compared with the normal epithelium of the Sox4 expression, in parallel with induction of p53 protein
gallbladder [110]. Reduced Sox4 expression is associated in medulloblastoma cell lines [113]. In addition, Sox4-
with high histological grade, high pathologic T stage, and knockdown dramatically blocks the radiation-induced
late clinical stage of this malignancy. In addition, expres- increases in p53 (S-15) phosphorylation and XRCC1 pro-
sion of Sox4 in tissues with positive nodal metastasis is tein expression along with an increase in levels of phospho-
lower than those without metastasis. In line with these c-H2AX compared with cells subjected to radiation alone
observations, Sox4 expression is positively correlated with [113], suggesting that Sox4 functions upstream of p53-
a better overall and disease-free patient survival [110]. It is mediated DNA repair, an important process in prevention
intriguing that, in the majority of studies that investigated of mutations and tumorigenesis.
the correlation between Sox4 expression and patient sur-
vival, Sox4 has been found as a positive marker for Role of Sox4/b-catenin axis in regulation
survival. For instance, despite an enhanced expression of of tumorigenesis
Sox4 in bladder carcinomas, there is a positive correlation
between strong Sox4 expression and increased patient Wnt signaling has long been implicated in tumorigenesis
survival [75]. Similarly, there is a trend toward favorable and deregulated expression of its components is a common
prognosis with increasing Sox4 expression levels in phenomenon in many types of tumors [114]. One of the
patients with medulloblastoma, despite the enhanced main mediators of the wnt signaling pathway is b-catenin,
expression of Sox4 in medulloblastoma compared with abnormal expression and/or mutation of which is a com-
normal cerebellum [111]. Also, Sox4 is overexpressed in mon feature of almost all types of human malignancies
hepatocellular carcinoma, but its expression correlates with [115]. In HEK293 cells, overexpression of Sox4 enhances
diminished risk of recurrence and improved overall sur- b-catenin/TCF activity by increasing the stability of
vival in HCC patients [77]. Nevertheless, none of these b-catenin, which induces wnt signaling pathway activity
studies addressed the observed discrepancy in the expres- and expression of its target genes, cyclin D1 and c-myc. The
sion level or function of Sox4 and its correlation with enhanced b-catenin/TCF activity by Sox4 is caused by
survival. In contrast to these reports that suggest Sox4 as a stabilization of the b-catenin protein, through induction of
marker for improved prognosis, at least in some cases Sox4 CK2, a kinase involved in regulation of b-catenin stability,
expression was found to be inversely correlated with sur- suggesting that Sox4 may act as an activator of the wnt
vival (Table 1). For instance, Sox4 expression inversely signaling pathway [116]. In a study that revealed a novel
correlates with overall patient survival of gastric cancer non-transcription related mechanism by which Sox4 may
[86] and recurrence-free survival of microsatellite-stable act as an oncogene, Sinner et al. [21] observed that, unlike
stage II (no lymph node or distant metastases) colorectal Sox17 that represses b-catenin/TCF activity and inhibits
carcinoma [112]. proliferation of SW480 colon carcinoma cells, Sox4
As mentioned earlier, in HCC cells, the HMG box enhances b-catenin/TCF activity and cell proliferation. In
domain of Sox4 interacted with p53, resulting in the inhi- fact, both Sox17 and Sox4 physically interact with TCF/
bition of p53-induced Bax expression and the p53- LEF family members via their HMG-box domains and
mediated apoptosis induced by gamma irradiation [77]. while Sox17 promotes their degradation, Sox4 stabilizes
However, it is not clear how Sox4 expression would confer b-catenin and TCF/LEF proteins [21].
a survival advantage to the patient while it can suppress the c-catenin, also known as junction plakoglobin (JUP), is
p53-mediated apoptosis. Also, the observed suppression of a Sox4 binding protein in prostate cancer cells [55].
p53 activity by Sox4 is in contrast to another report by Pan Interaction between these two proteins is enhanced by
et al. [20], who demonstrated that Sox4 induces p53 wnt3A treatment. However, this interaction could inhibit
activity in colon cancer cell lines. In these cells, expression Sox4 binding to downstream target promoters and may
of Sox4 protein but not mRNA is increased upon treatment repress its transcriptional activity in addition to modulation
with doxorubicin (DOX) and Sox4 is required for activa- of the b-catenin-mediated transcription. These observations
tion of p53 in response to DOX-induced DNA damage. In led to the suggestion that c-catenin may compete with
fact, in these cells, Sox4 interacts with and stabilizes p53 b-catenin for binding to Sox4, downregulating wnt-respon-
protein by repressing Mdm2-mediated ubiquitination and sive transcription [55]. This model is also consistent with
degradation of p53. Furthermore, Sox4 interacts with p300/ the observations that c-catenin expression is lost in human
CBP and enhances p300/CBP/p53 complex formation and prostate cancer through epigenetic and genetic pathways

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2690 S. M. Jafarnejad et al.

[117], as is its potential to suppress cell migration and which Sox4 contributes to the tumorigenesis (or suppres-
tumorigenic potential [118]. In malignant melanoma, Sox4 sion of tumorigenesis) in those cancers is not known. Also,
is reported to activate the wnt/b-catenin signaling pathway whereas differential and tissue-dependent regulation of
[119]. Accordingly, Sox4 knockdown leads to a decreased transcription is customary in Sox family, in some cases it is
b-catenin protein expression, resulting in inhibition of wnt/ not understood whether or not the mode of Sox4-mediated
b-catenin signaling pathway activity and reduced expres- regulation of activity, but not expression, of other proteins
sion of survivin as well as attenuated cell proliferation can also be tissue-dependent. For instance, in hepatocel-
[119]. However, In contrast to this observation, our own lular carcinoma cells, Sox4 interacts with p53, resulting in
studies have shown that knockdown of Sox4 slightly the inhibition of p53 activity and apoptosis [77], whereas,
increases melanoma cell growth [104]. in colon cancer cell lines, Sox4 interaction results in
As opposed to the aforementioned role of the Sox4/b- enhanced p53 activity and apoptosis [20]. Several members
catenin axis in the promotion of tumorigenesis, a recent of the Sox family have been implicated in tumorigenesis,
report has revealed that Sox4 could indeed enhance and in a number of cases some siblings compete with Sox4
b-catenin/TCF4 transcription, through upregulation of TCF4 in binding to and regulating the activity of the target pro-
at the transcription level, without any direct b-catenin moters or binding partners [16, 21, 36]. Nevertheless,
association, resulting in a significant decrease in the pro- except in a few studies, the possible roles of other Sox
liferation rate, along with increases in expression of p21, as proteins in Sox4 networks and their putative influence on
well as of TCF4, in endometrial carcinoma cells [53]. Sox4’s oncogenic or tumor suppressive functions remain
Consistently, Sox4-knockdown increased cell growth in unknown. Despite the recent progress in understanding the
this model. These observations provide evidence that at mechanisms which regulate expression of Sox4 (Fig. 2),
least in some types of malignancy the Sox4/b-catenin axis little is known about the mechanisms responsible for the
may contribute to suppression of tumorigenesis. In concert aberrant expression or function of Sox4 in cancers.
with this notion, although debated [120], b-catenin is In this review, we summarized the progress made over
implicated in suppression of melanoma invasion and more than two decades after the discovery of Sox4 in
tumorigenesis [121, 122]. In addition, at least in some cases defining its role in tumorigenesis, with an emphasis on the
expression of b-catenin is reduced during melanoma pro- tissue-dependent function of Sox4 as well as occasional
gression [123–125] and decreased b-catenin expression is discrepancies between various reports. Although the actual
linked to worsened patient survival [126, 127]. Considering reason for these discrepancies is not known, in some cases it
the loss of expression of Sox4 in melanoma and the con- may be due to the remarkable short half-life of the Sox4
siderable amount of evidence that Sox4 may regulate protein and the inability of mRNA expression studies to
b-catenin expression and/or activity, it would be interesting accurately reflect the level of protein expression. Indeed, it
to identify the possible role of Sox4 in regulation of seems that Sox4 mRNA expression may not be able to mirror
b-catenin in melanoma cells and its significance in tumor the expression level of its translation product [22], and is
progression. It should also be noted that in some cases insufficient to permit the drawing of a conclusion regarding
overexpression of Sox4 may suppress b-catenin expression. the expression levels of the Sox4 protein in different types of
In fact, overexpression of Sox4 induces a significant cancer. This notion is especially critical in the studies which
reduction of b-catenin expression in breast cancer cell lines use RT-PCR or cDNA microarray. It is worth emphasizing
[89], which further complicates the mutual relationship that Sox4 contains a single exon and, due to the lack of
between Sox4 and b-catenin and their functional outcome introns, it is difficult to distinguish between the sequences of
in different types of tissue. the genomic DNA and the complementary DNA (cDNA) in
studies that investigate the expression levels of the Sox4
transcript. Therefore, more precautionary measures such as
Concluding remarks DNase treatment of the extracted mRNAs are required to
avoid false positive signals due to genomic DNA contami-
In recent years, numerous publications have described the nation in these types of studies.
expression pattern and possible functional significance of At least one study has identified three variants, including
Sox4 in various types of malignancies. However, despite a nonsense mutation in the coding region of Sox4 that
this considerable progress, some outstanding questions could result in expression of a truncated isoform [76].
regarding the mechanistic details of Sox4 functionality and Although it is not known whether similar truncated iso-
its significance in cancer initiation and progression remain forms of Sox4 are also expressed in other types of tissues,
to be answered. For instance, in several types of cancer, the majority of studies regarding the expression status of
while the expression pattern of Sox4 is known to be dif- Sox4 in cancer did not consider its mutation status and
ferent than their normal counterparts, the mechanism(s) by possible influences of these mutations in stability and/or

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Role of Sox4 in tumorigenesis 2691

CK2 β-catenin

Proteasomal
degradation

DNA damage
MDM2
p53 p300/CBP

p53

β-catenin
p53 TCF
Sox2, Snail, CREB,
Dicer, Bax, etc

Oct4

Ets-1
Proteasomal Ubc9 γ-catenin Target promoters
degradation

p53
? IL-5, Sfpi1, Bax,
NF-κB p50, etc,.

Target promoter

Fig. 4 Regulation of gene expression by Sox4. So far, two modes of expression, which is discussed in the text, plays a very important role
regulation of gene expression by Sox4 have been identified; in oncogenic or tumor suppressive functions of Sox4. It should be
transcriptional and post-translational. At the transcriptional level, noted that not all of the illustrated complexes are present in every type
Sox4 can act both as a transcription factor and repressor, depending of cell
on the gene or cell type of interest. This differential regulation of gene

functional properties of the Sox4 protein in the tissues of cells, Sox4 can impede or augment activity of target pro-
interest. It is intuitive that, due to increased mortality moters. For instance, in melanoma cells, we observed that
associated with late stages of cancer, factors whose Sox4 inhibits NF-jB p50 [104] but induces Dicer [107]
expression positively correlates with tumor progression promoter activity. At the molecular level, this discrepancy
should have an inverse correlation with patient survival. might be explained by the requirement for specific partners
Nevertheless, in some cases, we observe a contradiction at the site of the target promoters for Sox4 to either
between Sox4 expression pattern and its correlation with enhance or inhibit their activity. So far, several factors,
survival (Table 1). It is plausible that these contradictions such as POU proteins Oct4 [44] and Brn2 [16], as well as
may be due to expression of multiple isoforms and/or other factors like Tbx3 [27], p53 [20], GATA-3 [32], TCF4
mutant forms of Sox4 in these malignant tissues, particu- [21], and JUP [55], have been identified to cooperate with
larly the mutants that lack the C-terminal domain and are Sox4 in the regulation of target promoters. These binding
more stable [76], perhaps due to the absence of the degron partners can have a crucial impact on the expression profile
located in this domain [22]. Future studies are required to of Sox4 downstream targets and its overall function in each
empirically validate the expression or lack of expression of type of cell (Fig. 4). The expression pattern of many of
such isoforms in other types of cancer and determine what these partners is differentially regulated in various cell
proportion (if any) of the Sox4 staining could be due to types or different developmental stages of the cells. For
these isoforms. instance, Oct4 is a well-known marker of pluripotency
At first glance, the inconsistency in the role of Sox4 in which is expressed in embryonic stem cells, some adult
various tumors might seem puzzling. Sox4 expression stem cells, and cancer progenitor cells, but not differenti-
gives a selective edge to some types of tumors, consistent ated cells [128]. This phenomenon would restrict the effect
with an oncogenic role, while it can suppress initiation or of Sox4 on the expression of those targets only to the cell
progression of other types of cancer, fitting to the definition types that express both Sox4 and Oct4. Nevertheless, in
of a tumor suppressor. As mentioned earlier, this feature is most cases, the putative binding partners of Sox4 are not
characteristic of Sox family whose function relies on the known, which beckons further attention in future studies to
context of the host cells. In addition, in the same type of this important aspect of the Sox4 network.

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Sox4 regulates the expression of a large set of genes in modulation of Sox4 in one tissue may also end up as a
different tissues as evidenced by genome-wide cDNA major side effect in another type of tissue.
microarray or promoter binding profiling studies. Despite
this large number, there seems to be very limited overlap Acknowledgments This work was supported by Canadian Institutes
of Health Research (MOP-84559, MOP-93810 and MOP-110974),
between the Sox4 targets identified in different studies Canadian Cancer Society Research Institute (2011-700714) and
(Fig. 3). This negligible overlap further indicates the Canadian Dermatology Foundation to G.L. S.M.J and R.S.A. are
existence of distinct Sox4 downstream pathways in each recipients of the trainee award from Canadian Institute of Health
cell type. Also, in the majority of cases, it is not clear Research Skin Research Training Centre. S.M.J. is a recipient of
Roman M. Babicki Fellowship and the University of British
whether regulation of target gene expression by Sox4 is Columbia Graduate Fellowship.
achieved by direct binding to their promoters and acting as
a transcription factor or via indirect modes such as acti-
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