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REVIEW ARTICLE
Sp1 and the ‘hallmarks of cancer’Kate Beishline and Jane Azizkhan-Clifford
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA, USA
Keywords
cancer; oncogene; Sp1; transcription; tumor
suppressor
Correspondence
J. Azizkhan-Clifford, Department
Biochemistry and Molecular Biology, Drexel
University College of Medicine, 245 North
15th Street, MS 497, Philadelphia, PA
19102, USA
Fax: 214 762 4452
Tel: 215 762 4446
E–mail: Jane.Clifford@DrexelMed.edu
(Received 23 April 2014, revised 26
September 2014, accepted 10 November
2014)
doi:10.1111/febs.13148
For many years, transcription factor Sp1 was viewed as a basal transcrip-
tion factor and relegated to a role in the regulation of so-called housekeep-
ing genes. Identification of Sp1’s role in recruiting the general transcription
machinery in the absence of a TATA box increased its importance in gene
regulation, particularly in light of recent estimates that the majority of
mammalian genes lack a TATA box. In this review, we briefly consider the
history of Sp1, the founding member of the Sp family of transcription fac-
tors. We review the evidence suggesting that Sp1 is highly regulated by
post-translational modifications that positively and negatively affect the
activity of Sp1 on a wide array of genes. Sp1 is over-expressed in many
cancers and is associated with poor prognosis. Targeting Sp1 in cancer
treatment has been suggested; however, our review of the literature on the
role of Sp1 in the regulation of genes that contribute to the ‘hallmarks of
cancer’ illustrates the extreme complexity of Sp1 functions. Sp1 both acti-
vates and suppresses the expression of a number of essential oncogenes
and tumor suppressors, as well as genes involved in essential cellular func-
tions, including proliferation, differentiation, the DNA damage response,
apoptosis, senescence and angiogenesis. Sp1 is also implicated in inflamma-
tion and genomic instability, as well as epigenetic silencing. Given the
apparently opposing effects of Sp1, a more complete understanding of the
function of Sp1 in cancer is required to validate its potential as a therapeu-
tic target.
Introduction
The transcription factor Specificity Protein 1 (Sp1) was
first identified as a promoter-specific binding factor
that is essential for transcription of the SV40 major
Immediate Early (IE) gene [1,2]. Initially, Sp1 was
considered a general transcription factor that is
required for transcription of a large number of ‘house-
keeping genes’, so-called because of their involvement
in metabolism, cell proliferation/growth, and cell death
[3]. It has become increasingly clear that many of the
housekeeping genes play critical roles in cancer initia-
tion and progression. Estimates suggest that there are
at least 12 000 Sp binding sites within the human gen-
ome, and most studies support the idea that Sp1 not
only maintains basal transcription, but also contributes
to the regulation, i.e. induction and inhibition, of tran-
scription of a large number of cellular genes [4–6]. The
Abbreviations
Akt, protien kinase B; ATM, ataxia telangiectasia mutated; ATR, AT and Rad3 related; Brca1, breast cancer associated factor 1; b-TCRP,
beta-transducin containing protein; EGFR, epidermal growth factor receptor; ER, estrogen receptor; ERK, extracellular signal-regulated
kinase; GSK3b, glycogen synthase kinase-3 beta; IGF, insulin-like growth factor; IGF1R, insulin-like growth factor receptor 1; JNK1, Jun N-
terminal kinase 1; MMPs, matrix metallproteinases; NSAIDS, non-steroidal anti-inflammatory drugs; PI3K, phosphoinositide 3-kinase; PKC,
protein kinase C; PML, promyelocytic leukemia protein; ROS, reactive oxygen species; SCF, Skp–Cullin–F box; VEGF, vascular endothelial
growth factor; VHL, Von Hippel-Lindau Protein.
224 FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
activities of Sp1 are regulated throughout the cell
cycle, and are modulated by post-translational modifi-
cation(s) in response to a large array of signals as dis-
cussed here. We also discuss the regulatory functions
of Sp1, as well as key protein–protein interactions,
with a focus on its role in cancer progression.
Sp1 was the founding member of the Sp transcrip-
tion factor family, the members of which contain
C2H2-type zinc fingers and resemble the larger family
of ‘Kr€uppel-like factors’ [3,7,8]. The Sp factors prefer-
entially bind GC boxes, while Kr€uppel-like factors
bind CACCC boxes [9–14]. Sp factors may be sepa-
rated into two groups: Sp1–4 and Sp5–9. The domain
organization of Sp1–4 is very similar, while Sp5–9 are
significantly different from Sp1–4, and are more simi-
lar to the other Kr€uppel-like factors. Sp1–4 have simi-
lar N–terminal transactivation domains (Fig. 1)
characterized by glutamine-rich regions, which, in
most cases, have adjacent serine/threonine-rich regions.
There is also a highly charged region referred to as the
C domain. Although the transactivation domains are
similar in overall amino acid content, they are not sig-
nificantly homologous to one another, which accounts,
at least in part, for differences in their functions. The
DNA-binding domains of Sp1–4 include three highly
homologous C2H2-type zinc fingers, which preferen-
tially bind to the same GC consensus site [50-(G/T)
GGGCGG(G/A)(G/A)(G/T)-30] [15,16]. Each of three
zinc fingers in Sp1 has its own specific sequence
preference, and all three are required for high-affinity
binding, with the first zinc finger having the highest
sequence specificity [17,18]. Interestingly, loss of all
three Sp1 zinc fingers abolishes not only DNA binding
but also nuclear localization. Nuclear localization
requires coordinated binding of zinc [19,20].
In contrast to other Sp family members, Sp1 con-
tains a C–terminal multimerization domain that medi-
ates super-activation of promoters containing multiple
adjacent Sp sites. Here, DNA-bound and unbound
Sp1 molecules interact, forming tetramers that develop
into larger multimers that promote association of
proximal promoter regions with distal enhancers by
forming looped regions of DNA [21,22]. This super-
activation mechanism is discussed further below. Mul-
timerization also exposes various regions of the Sp1
molecules for protein–protein interactions and post-
translational modifications.
Sp3 is the family member that is most similar to
Sp1. Unlike Sp1, Sp3 does not contain a multimeriza-
tion domain. Instead, it contains an inhibitory domain
located just N–terminal to its DNA-binding domain,
that mediates transcriptional repression in some
instances [23–25]. The D domain of Sp3, which bears
little similarity to the D domain of Sp1, may some-
times act as a transcriptional inhibitor [26]. Sp1 and
Sp3 have equal affinity for the GC box binding motifs,
and, as such, their relative abundance and binding
ratios influence gene regulation [27]. All these data are
consistent with the notion that the presence of a single
Sp site allows binding of either Sp1 or Sp3 to promote
Fig. 1. Transcription factor Sp1 and its closest related family members. Sp1 and its four closest family members are grouped together
based on their Cys2His2 zinc finger DNA binding domains (purple) and transactivation domains (light and dark blue), which are located N–
terminal to the DNA binding domain. The transactivation domains are composed of serine/threonine-rich regions, flanked by regions rich in
glutamine. The charged domain (green), although not essential for DNA binding, promotes binding of the zinc fingers to their DNA target.
Only Sp1 contains a multimerization domain (beige) which facilitates interaction between multiple Sp1 molecules and promotes super-
activation of genes. The position of the Buttonhead domain (Btd), which is conserved in Drosophila Sp1 and the similar Drosophila protein
Buttonhead, is indicated. Grey regions lack similarity between proteins.
225FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
K. Beishline and J. Azizkhan-Clifford Sp1 and the hallmarks of cancer
basal transcription. Unlike Sp1, Sp3 does not have the
ability to super-activate promoters by bridging multi-
ple Sp binding sites. This finding supports the possibil-
ity of a potential inhibitory function for Sp3 in
promoters containing multiple Sp sites; in regulatory
regions with multiple sites, the presence of Sp3 at one
or more of these sites may interfere with Sp1 binding
and suppress the gene activation that requires multi-
merization between multiple Sp1 molecules on adjacent
or distal binding sites [23,24]. An example of this phe-
nomenon is regulation of the human topoisomer-
ase IIa promoter, for which activation occurs by
bridging between Sp1 bound at sites in the distal
enhancer and the proximal promoter. Here, proximal
and distal binding of Sp1 activates transcription, while
competition between Sp1 and Sp3 for binding at either
the distal enhancer or both binding regions results in
Sp3-dependent repression [28]. Displacement of a sin-
gle Sp1 molecule by Sp3 leads to a decrease in Sp1-
dependent transactivation [24,29]. The interplay
between Sp1 and Sp3 is discussed further below.
Both Sp1 and Sp3 are essential for embryonic devel-
opment, and knockout of either factor is embryo-lethal
in mice [30–32]. It has also been shown that Sp1 and
Sp3 are differentially regulated during development
[33], implying a role for these factors in cellular differ-
entiation and development.
Sp1 is a eukaryotic specific factor, and the full-
length Sp1 protein is highly conserved among mamma-
lian species. The family is defined by the DNA-binding
domain, which is also conserved in fish, reptiles and
birds. There are multiple Sp/Kr€uppel-like factor family
members that regulate various stages of development;
there is a high degree of amino acid conservation, par-
ticularly in the zinc-finger region, between Drosophila
Sp1 and those of higher species [34]. As indicated in
Fig. 1, Sp1 also contains a small region of homology
with the Drosophila protein buttonhead [35]. A yeast
homolog of any of the Sp1 factors has yet to be identi-
fied based on sequence homology or conservation of
its DNA binding ability. Moreover, no functional
homolog has been found either.
Sp1 contains two main transactivation domains, A
and B, which, in cooperation with the DNA binding
domain, support the majority of Sp1’s transcriptional
activity (Fig. 1). Domains A and B are highly gluta-
mine- and serine/threonine-rich, with domain B being
over twice as lard as domain A [36]. These transactiva-
tion domains have no sequence-specific transcriptional
activity alone, and require binding to promoters
through the DNA-binding domain to promote tran-
scription [37]. The C domain is highly charged, and
supports both DNA binding and transactivation,
although it is not essential for either process [37]. The
D domain, as discussed above, is important for multi-
merization, but other portions of the protein probably
also participate in this process.
Domains A, B, C and D are largely unstructured,
containing no known structural elements. In fact, the
only structured region of Sp1 is the zinc-finger domain,
for which an NMR structure has been elucidated
[38,39]. The highly disordered structure of the protein
suggests that Sp1 is probably capable of interacting with
a large number of protein partners, as discussed below.
Sp1 has three isoforms. Isoform A is the primary
form of the protein, containing 785 amino acids. Iso-
form B is a translational variant, in which an alterna-
tive start codon is used, resulting in a protein lacking
the first seven amino acids. Some studies, discussed
below, have suggested that these amino acids affect
the stability of the protein. The final variant, isoform
C, results from use of an alternative splice acceptor
site in the 3rd exon of the Sp1 pre-mRNA, which pro-
duces a protein that lacks amino acids 55–102 [40].
Few papers have discussed the functional significance
of the various isoforms, and more work is required to
identify the potentially different expression and func-
tions of each isoform.
Regulation of Sp1
As suggested by early publications [41,42], Sp1 is
highly modified by almost all the common forms of
post-translational modification, including phosphory-
lation, O-linked glycosylation, acetylation, SUMOyla-
tion, and ubiquitylation, and is targeted to
proteasome-mediated degradation pathways. Table 1
lists the post-translational modifications reported in
the literature, and their effect on Sp1 activity (when
documented).
Post-translation modifications
The 785 amino acid Sp1 protein contains 164 serine
and threonine residues, suggesting that it may be
highly phosphorylated and O-glycosylated. These
modifications affect not only Sp1 activity, but also
its stability, through modulation of proteolytic cleav-
age and proteasomal degradation. Several of the
modifications that have been identified in vitro or
through screens have yet to be associated with spe-
cific functions in cells. Signaling through specific kin-
ases probably has effects that have yet to be
discovered. Several of these modifications are dis-
cussed below within the context of modulating Sp1
stability and transcriptional activity.
226 FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
Sp1 and the hallmarks of cancer K. Beishline and J. Azizkhan-Clifford
A number of key signaling kinases have been shown
in vitro and in vivo to both directly and indirectly mod-
ify Sp1. These include extracellular signal-regulated
protein kinases (ERK) 1 and 2, c–Jun N–terminal
kinase 1 (JNK1), cyclin-dependent kinase 2 (CDK2)
and the phosphatidylinositol 3–kinase (PI3K)-like
kinases, ataxia telangiectasia mutated (ATM), ataxia tel-
angiectasia and Rad3-related kinase (ATR) and DNA-
dependent Protein Kinase (DNA-PK) (see Table 1 for
extensive list with references). These phosphorylation
events contribute to further post-translational modifi-
cation of Sp1, modulation of Sp1 stability, Sp1 DNA
binding affinity, and Sp1’s ability to transactivate tran-
scription. A number of serine/threonine residues have
been shown to be both phosphorylated and glycosylated,
and the dynamic between these two modifications is
probably important for regulation of function.
A single Sp1 molecule has the potential to be
extensively modified. The number of possible combina-
tions of modifications suggests that Sp1 may be
differentially modified even within the same cell, with
various levels of stability and differential functions
depending on the combination of modifications. As
mentioned above, the majority of the Sp1 protein is
unstructured, based on primary sequence information
[74], consistent with the idea that post-translational
modifications may modulate its structure. These modi-
fications and associated structural alterations support
the ability of Sp1 to interact with a large number of
binding partners, and to differentially modulate tran-
scription of a large number of genes.
Regulation of Sp1 stability
The levels of Sp1 protein are tightly regulated to main-
tain cellular homeostasis. Sp1 levels are modulated
throughout the cell cycle, and transcriptional regulation
is in part achieved by variable protein level. An early
study implicated reduced Sp1 O–glycosylation, associ-ated with glucose starvation, with its degradation [75].
Table 1. Sp1 post-translation modifications.
Residue Modification Enzyme Function References
Ser2 Phosphorylation; N–acetylserine Unknown Unknown [43,44]
Ser7 Phosphorylation Unknown Stability [43–45]
Lys16 SUMOylation; ubiquitinylation RNF4 Stability [45–48]
Ser56 Phosphorylation ATM/ATR Unknown [49]
Ser59 Phosphorylation CDK2; ERK1/2; PP2A Stability, DNA binding [45,50–53]
Ser101 Phosphorylation ATM/ATR – [49,54,55]
Ser111-114 Glycosylation OGT – Unpublishedb
Asp183 Cleavage Casp3 Unknown Unpublishedc
Ser220a Phosphorylation DNA-PK Transcription [56]
Thr278 Phosphorylation JNK1; ERK1/2 Stability [47]
Ser301 Glycosylation OGT – Unpublishedb
Thr355a Phosphorylation ERK1/2; JNK1 Transcription [57]
Thr453 Phosphorylation ERK1/2 Transcription [58–60]
Ser491 Glycosylation OGT Transcription [61,62]
Ser535/540 Glycosylation OGT – Unpublishedb
Asp584 Cleavage Casp3 – [63]
Ser612 Phosphorylation; glycosylation OGT Localization [64]
Thr640 Phosphorylation; glycosylation OGT Localization [64]
Ser641 Phosphorylation; glycosylation PKCf; OGT Transcription [64,65]
Thr651 Phosphorylation PKCf [66]
Thr668a Phosphorylation CKII; PPI; PKCf DNA binding [64,67,68]
Ser670 Phosphorylation PKCf – [68]
Thr681 Phosphorylation PP2A; PKCf – [50,68]
Ser698 Glycosylation Localization [64]
Ser702 Glycosylation OGT – [64]
Lys703 Acetylation P300; HDAC1 – [69–72]
Ser728 Phosphorylation GSK3b Degradation [73]
Ser732 Phosphorylation GSK3b Degradation [73]
Thr739 Phosphorylation Erk1/2, JNK1 Transcription, stability [47,48,58,73]
a Amino acid residue number is based on the full length 785 aa Sp1 protein. b Unpublished data from in vitro glycosylation assays Beishline
K, Azizkhan-Clifford J. c Unpulished data Torabi B, Azizkhan-Clifford J. Paranthetic Numbers indicate amino acid number in original Sp1
sequence which lacks amino acids 1–89.
227FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
K. Beishline and J. Azizkhan-Clifford Sp1 and the hallmarks of cancer
The degradation occurred as a result of a cleavage event
in the N–terminus of the protein, which was dependent
on the presence of the first seven amino acids [75,76],
and was suggested to occur via interaction with the pro-
teasome [77]. Sp1 was also shown to interact directly
with b–transducin repeat-containing protein (b–TCRP),
a component of the Skp–Cullin–F box (SCF) ubiquitin
ligase complex [73], further supporting an interaction
with the proteasome. b–TCRP probably interacts with
Sp1 through a DSG (Asp-Ser-Gly) destruction box (b–TCRP binding motif) within the C–terminus of Sp1
[DSGAGS(727–732)], mediating proteasomal degrada-
tion of the protein. Evidence suggests that ubiquitin-
mediated degradation may be modulated by phosphor-
ylation of Sp1 by both ERK (on threonine 739) and
Glycogen synthase kinase-3 beta (GSK3b) (on threo-
nine residues 728 and 732) [73]. Despite strong evidence
for ubiquitin-mediated proteasomal degradation of
Sp1, residue(s) on Sp1 that are directly ubiquitylated
have yet to be identified.
The stability of Sp1 is also affected by SUMOyla-
tion, which occurs on lysine 16. This modification is
modulated by surrounding residues, and requires the
first seven amino acids of Sp1, which were previously
found to be associated with protease-mediated cleav-
age [45]. SUMOylation was shown to be associated
with destabilization of Sp1 [45,46,78], and later studies
showed that destabilization occurs through Ring finger
protein 4 (RNF4), a SUMO-dependent E3 ubiquitin
ligase. RNF4 interacts with both the SUMO-modified
region of Sp1 and its DNA-binding domain. This
interaction is associated with RNF4-dependent ubiqui-
tylation of Sp1, which is dependent on Sp1 SUMOyla-
tion [48]. The association of RNF4 with the DNA-
binding domain of Sp1 also suggested that RNF4 may
potentially affect Sp1 function independently of Sp1
ubiquitylation, and that Sp1 may modulate interaction
between RNF4 and other targets.
Phosphorylation of Sp1 modulates proteasome-
mediated degradation during mitosis. JNK1 phos-
phorylates Sp1 on threonine residues 278 and 739,
and these modifications are associated with stabiliza-
tion of Sp1 during mitosis [47]. Furthermore, JNK1-
dependent phosphorylation of threonine 739 inhibits
the interaction between Sp1 and RNF4, supporting
a role for JNK1 in promoting Sp1 stability [48]. The
effects of phosphorylation of threonine 278 on the
interactions between Sp1 and RNF4 or Sp1 and b–TCRP are unclear. This suggests the need for further
studies to address how Sp1 phosphorylation affects
SUMOylation and ubiquitylation, and the possibility
that Sp1 may be targeted by multiple E3 ubiquitin
ligase complexes.
Protein–protein interactions involving Sp1
Sp1 has been shown to interact with a number of cel-
lular factors to modulate expression of specific genes,
facilitate post-translational modification of Sp1, and
regulate Sp1 stability. Table 2 provides a comprehen-
sive list of factors that have been shown to interact
with Sp1. These include members of the basal
transcriptional machinery, other transcription factors,
cell-cycle regulators, chromatin modifiers and ATP-
dependent remodelers, as well as factors participating
in cellular processes such as DNA repair. A number of
these interactions are associated with specific cellular
functions, which are discussed in more detail through-
out later sections. A number of factors listed in table 2
have only been shown to interact in vitro [117] and
many have not been associated with specific cellular
functions. Sp1 may interact with multiple factors
simultaneously, particularly when present as a multi-
mer, resulting in a large number of complexes. This
makes it difficult to distinguish specific Sp1 functions
within a single complex, and also makes identifying
direct Sp1 interactions in vivo experimentally difficult.
Sp1 in cancer
While often described as a general transcription factor,
Sp1-dependent transcription is highly regulated
throughout development, cellular differentiation and
tumorigenesis. Sp1 differentially regulates a large num-
ber of key factors that are important in a number of
disease states, including cancer, which is the focus of
the remainder of this review.
Sp1 is over-expressed in a number of cancers,
including breast, gastric, pancreatic, lung, brain (gli-
oma) and thyroid cancers (Table 3) [156–160]. In
patient samples and cancer models, Sp1 levels correlate
with stage, invasive potential and metastasis. Sp1 levels
correlate with the survival of patients in almost all
cancers, with high levels of Sp1 being associated with
poor prognoses. On this basis, a number of studies
have addressed the possibility of targeting Sp1 to treat
cancer cells or sensitize tumors to other treatments
[161–166]. However, specific inhibitors that target Sp1
have not been developed, and the feasibility of target-
ing Sp1 specifically in cancer treatment has not been
demonstrated. This is at least in part due to the myr-
iad activities of Sp1 and incomplete mechanistic under-
standing of Sp1 function.
Hanahan and Weinberg have thoroughly reviewed
the cellular pathways essential for tumor formation
and cancer progression [170,171]. These include the
eight major hallmarks of cancer: sustained proliferative
228 FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
Sp1 and the hallmarks of cancer K. Beishline and J. Azizkhan-Clifford
Table 2. Sp1 interactions.
Protein Region References
General transcription factors
TBP A, B [79,80]
TAF4 A, B [61,83–89]
TAF7 Unknown [95]
TFIIAa Unknown [96]
dTAFII110 Unknown [61,62]
TAF–Ia/b Unknown [99]
Transcription factors
p53 Unknown [106,107]
E2F1 Amino acids 699–785 [102,110]
c–MYC Unknown [111]
c–Jun Amino acids 424–542 [113]
TReP–132 Unknown [114]
C/EBPb Unknown [116]
PML Unknown [118]
ELF1 Unknown [120]
Oct1 Unknown [124]
NF–YA Unknown [125]
Che–1 Unknown [126]
EGR1 Unknown [127]
DLX4 Unknown [128]
HIF1a Unknown [129]
YYI Unknown [131]
Fli–1 Unknown [132]
SREBP2 Unknown [133,134]
SMAD2 Unknown [135,136]
STAT6 Unknown [138]
Other factors
TG2 Unknown [140,141]
Huntingtin Unknown [142]
HIV–1 Tat Unknown [56,144]
RNF4 Amino acids 619–785 [48]
PKCf Unknown [121]
Chromatin remodeling factors
p300 Amino acids 138–232
Amino acids 262–487
[69,71,81,82]
SWI/SNF complex, BRG1, BAF155, BAF170 Unknown [90–94]
HMGA1 Unknown [97]
HMGA2 Unknown [98]
DNMT1 Unknown [100,101]
HDAC1 Amino acids 622–788 [69,102–105]
HDAC2 Unknown [103,108,109]
RbAp48 Unknown [103]
MCAF1/2 Unknown [112]
Tumor suppressors
Brca1 Unknown [115]
SKP2 Unknown [117]
MDM2 Unknown [119]
VHL Unknown [121–123]
b–TCRP Unknown [73]
DNA repair factors
Nbs1 Unknown [74]
Brca2 Unknown [117]
Rad51 Unknown [117]
ATM Unknown [130]
229FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
K. Beishline and J. Azizkhan-Clifford Sp1 and the hallmarks of cancer
signaling, replicative immortality, resistance to cell
death and avoidance of immune destruction, induction
of angiogenesis, invasion and metastasis, and de-regu-
lation of cellular energetics [170,171]. Sp1 is critical for
the regulation of genes whose products are responsible
for each of these hallmarks. In addition, Sp1 may
influence two of the ‘enabling characteristics’ of can-
cer: inflammatory signaling, which alters the oxidative
state of the cell [170], and maintenance of genomic sta-
bility. Here we discuss key genes for which Sp1 has
been shown to play a regulatory role specifically in
tumors (Table 4).
Sp1 modulates cancer cell proliferative and
survival signals
Sustained proliferative signaling: IGF1R
The requirement for cancer cells to maintain their own
proliferative signals, independent of normal physiologi-
cal signaling, is well established. A large number of
essential signaling molecules in normal and cancer cells
are associated with Sp1-mediated transcriptional regu-
lation, including epidermal growth factor and its
receptor, fibroblast growth factor, and insulin-like
growth factor and its receptor (Table 4). In addition,
growth signaling directly regulates Sp1 and additional
Sp1-dependent proliferative signals.
One of the best examples of an Sp1-regulated
growth signaling molecule that is disrupted in cancer is
insulin-like growth factor 1 receptor (IGF1R). It is
well established that insulin-like growth factor (IGF)
signaling is utilized by cancer cells to maintain the pro-
liferative program, and, for this reason, IGF1R has
been proposed as a potential target in tumor treatment
[300,301]. When the IGF1R promoter was first charac-
terized, it was shown to contain as many as eight Sp
target sequences [195], and subsequent work showed
that a specific Sp1 site is essential for IGF1R transcrip-
tional regulation (Fig. 2) [213]. It is now understood
that growth signals directed through insulin-responsive
binding protein and Sp1 coordinate binding to GC-
rich and AT-rich regions of the IGF1R promoter to
increase transcription [302]. Further evidence suggests
that additional factors, including breast cancer-associ-
ated factor 1 (BRCA1), high-mobility group A1, estro-
gen receptor a (ERa) and ATM kinase, associate with
Sp1 (see Table 2) and modulate IGF1R transcription
[97,198,201].
BRCA1 has been shown to play a role in suppress-
ing Sp1-mediated regulation of IGF1R, which has sig-
nificant implications for the development and
Table 2. (Continued).
Protein Region References
Cell cycle
p21 Unknown [117]
CDK4 Unknown [117]
Cyclin A Unknown [137]
Nuclear receptors
SMRT N–terminus [139]
NCoR/BCoR N–terminus [139]
PPARc Unknown [143]
AR Unknown [145]
ERa Unknown [116,146–155]
Table 3. Sp1 expression in cancer.
Cancer
type References
Sp1
expression Survival Invasiveness, proliferation
Gastric [167] High Decrease –
Diffuse-type gastric [168] High Decrease Positive
Intestinal-type gastric [168] Low Decrease Positive/negative
Pancreatic [158] High Decrease Positive
Lung, early [169] High – –
Lung, stage IV [169] Low – Negative
Breast [160] High Decrease Positive
Glioma [159] High Decrease –
Thyroid [156] High – –
230 FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
Sp1 and the hallmarks of cancer K. Beishline and J. Azizkhan-Clifford
progression of breast cancer in carriers of the familial
BRCA mutation(s). Over several years, the Werner
group have established that BRCA1 negatively
regulates IGF1R transcription through direct interac-
tion with Sp1 that inhibits Sp1 binding at the IGF1R
promoter [199,200,262]. The interaction between Brca1
and Sp1 is stimulated by IGF1 [199,262]. BRCA1
directly interacts with Sp1 through amino acids 260–802, but the region of Sp1 that binds Brca1 has yet to
be identified [115]. It was later established that individ-
uals with the BRCA1 mutation have increased levels
of IGF1R compared to patients lacking the BRCA1
mutation [199]. This suggests that individuals with the
BRCA1 mutation are probably able to maintain
increased IGF1R transcription in the presence or
absence of IGF1 stimulation, due to a lack of Sp1-
dependent transcriptional inhibition.
EGFR
The epidermal growth factor receptor (EGFR) and its
family of tyrosine kinase receptors are associated with
sustained proliferation, as well as promoting invasion
and metastasis in cancer cells. Mutations in EGFR
have been identified in a number of cancers, and, in
addition, the levels of receptor are modulated to pro-
mote increased signaling in tumor tissues. For these
reasons, EGFR and its family of receptors have
Table 4. Cancer-related genes regulated by Sp1.
Gene References
Sustained proliferation/immortality
hTERT/hTERC [98,105,108,172–181]
p53/MDM2 [182–187]
p16 [188–190]
p21 [191–194]
IGF1R [97,106,115,195–201]
EGFR [202–207]
EGF [208]
FGF [209–212]
IGF [213–218]
Apoptosis
Survivin [162,164,172,173,219–224]
Trail–R2 [163,225,226]
Bcl–2 [70,227]
TRAIL [228]
MCL–1 [221,229]
XIAP [230]
Bak [70,168]
FasL [219,231–233]
Angiogenesis
VEGF [59,121,166,167,234–252]
TSP–1 [253]
PDGF [58,123,254–257]
uPA [258–260]
DNA damage/stress response
Brca1 [261,262]
ATM [130,263,264]
MDC1 [265]
Cdc25B [266,267]
RECQ4 [268]
PARP [269,270]
XRCC1 [271,272]
BLM [273]
CHEK2 [274,275]
XPC [276]
XPB [277,278]
XPD [277]
DDB1/2 [279]
DNA–PK/Ku70/80 [280]
XRCC5 [281]
ERCC6 (CSB) [282]
WRN [283]
Invasion and metastasis
MMP9 [60,284]
MT1–MMP [285,286]
RECK [60,287–289]
E–cadherin [169,290–294]
Integrin a5 [153,295,296]
MMP2 [90,137,159,284,297–299]
Fig. 2. Organization of GC boxes in Sp1-regulated promoters. Each
Sp1-regulated gene has a unique organization of transcription factor
binding sites. Multiple sites have been identified as being key for
Sp1-dependent increases or decreases in transcriptional activity of
the genes. It should be noted that the indicated enhancing and
inhibitory effects may be due to both binding or lack of binding at
these sites, depending on the gene context. Sp1 sites may be
found both upstream and downstream of transcription start sites,
and may coordinate with nearby binding sites for other factors. In
addition, other Sp factors, such as Sp3, may bind the same sites
and have different regulatory effects.
231FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
K. Beishline and J. Azizkhan-Clifford Sp1 and the hallmarks of cancer
become targets for chemotherapeutic intervention in
recent years [303].
Sp1 was identified as a transcriptional regulator of
EGFR in the late 1980s, and Sp1 probably participates
in regulation of EGFR in cancer [202,204,205]. Upon
further assessment, a number of factors have been
shown to either inhibit or activate Sp1-dependent tran-
scription of the EGFR gene. Pro-myelocytic leukemia
protein (PML) has been shown to associate with Sp1,
and this interaction inhibits transcriptional activation
of the EGFR promoter. The interaction occurs through
the dimerization domain of PML and the DNA-bind-
ing domain of Sp1, thereby inhibiting Sp1’s ability to
bind to its target sequences within the EGFR promoter
and probably other promoter regions [118]. More
recent studies have shown that over-expression of
PML enhances localization of Sp1 to PML nuclear
bodies, and increases Sp1 degradation [304]. SUMOy-
lation of Sp1 was found to promote its localization to
PML nuclear bodies and subsequent Sp1 degradation.
Degradation of SUMOylated Sp1 has been associated
with RNF4, and is inhibited by phosphorylation
events during mitosis, when it has been suggested that
Sp1 is generally evicted from chromatin [305]. It may
be interesting to determine whether there is overlap
between the PML-mediated pathway and other aspects
of SUMO-mediated regulation of Sp1 throughout the
cell cycle [45,46,78].
Other factors that regulate EGFR in an Sp1-depen-
dent manner include the histone deacetylase HDAC1,
which has been shown to bind in a complex with
Kruppel-like factor 10 (KLF10/TIEG1) to Sp1 sites
within the promoter and to inhibit histone acetylation
to suppress transcription [306]. In addition, the reti-
noic acid receptor has been shown to bind Sp1 sites
within the EGFR promoter and inhibit Sp1 binding to
its target sequences and suppress transactivation [307].
This is in contrast to estrogen receptors a and b, bothof which have been shown to transactivate the EGFR
promoter through interaction with Sp1 rather than
through direct binding [206,308].
Enabling replicative immortality
It is well established that normal cells have finite repli-
cative potential. Cells gradually approach their replica-
tive limit until they enter a senescent state. A cell that
is capable of overcoming these proliferative barriers
becomes immortalized (in culture), and may become
tumorigenic (in vivo). Cellular senescence requires a
number of factors, but primarily involves the CDK
inhibitor p16(Ink4a), transcription factor p53, and sta-
bilization of telomeres at the ends of chromosomes.
Sp1 is an essential regulator of the genes encoding
p16, p53 and several components of telomere mainte-
nance, including telomerase (Table 4), making it an
essential factor to evade entrance into senescence and
achieve replicative immortality.
p53
Like Sp1, p53 is a transcription factor that is impli-
cated in the regulation of a large number of genes.
There is a wealth of data available regarding p53’s role
in tumorgenesis, suggesting that the dynamics between
Sp1 and p53 are key to understanding many tumor-
suppressing pathways. A number of these pathways
are highlighted here, demonstrating strong cooperation
between Sp1 and p53 in the regulation of many tumor
suppressors and oncoproteins, including p21, IGF1R,
BCL2 binding component 3 (PUMA), DNA (cytosine-
5) methyltransferase 1 (DMNT1) and others
[106,107,309,310]. It is important to note that regula-
tion of transcription by p53 and Sp1 is dynamic and
variable among genes. In some instances, the proteins
work together to increase gene transcription [100,107],
while other studies show opposing effects on transcrip-
tion of certain factors [283,311]. In-depth studies are
required to fully appreciate the significance of Sp1/p53
gene regulation.
Independent of the transcriptional activities of these
two factors together, Sp1 is implicated in regulation of
MDM2 proto-oncogene, E3 ubiquitin protein ligase
(MDM2), the key regulator of p53 stability in cancer
cells. A number of small nucleotide polymorphisms in
the MDM2 promoter have been shown to be associ-
ated with increased expression of the protein due to
enhanced transactivation by Sp1 [182–187]. The conse-
quence of this enhanced MDM2 expression is down-
regulation of p53 and increased cancer susceptibility.
Although there is some disagreement regarding the sig-
nificance of these small nucleotide polymorphisms
[312], other factors such as EGFR contain small nucle-
otide polymorphisms that are associated with increased
Sp1-dependent transcription, which affects the cellular
signaling environment to favor tumorigenesis [313].
This suggests a mechanism whereby Sp1 directly mod-
ulates cancer signaling, through genetic mutations that
directly enhance the ability of Sp1 to induce tumor-
promoting transcriptional changes.
p16
As mentioned above, the CDK inhibitor p16 is a key
regulator of cellular senescence. In normal dividing
cells, p16 functions to inhibit cyclin CDK4/6 activation
232 FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
Sp1 and the hallmarks of cancer K. Beishline and J. Azizkhan-Clifford
of the retinoblastoma protein, a key tumor suppressor
protein that controls cellular proliferation in the G1
phase [314]. p16 has been found to be either mutated or
silenced in a large number of malignancies in multiple
tissues [315]. However, evidence suggests that p16 may
be over-expressed in some cancers [316]. How p16 func-
tions in oncogenic signaling is still unclear [316], despite
its certain role in tumor formation and progression.
Sp1 is a key regulator of p16 expression [188–190].The proximal promoter of p16, a region essential for
p16 expression, contains multiple Sp sites (Fig. 2), and
expression of Sp1 is important for maintaining pro-
moter activity [189]. The p16 proximal promoter must
also be modified by p300, a histone acetyl transferase,
to increase H4 acetylation levels and allow for tran-
scriptional activation. p300 interacts directly with the
transactivation domains of Sp1, and is recruited to the
p16 promoter [188]. Sp1 was shown to be important
for the up-regulation of p16 in normal human fibro-
blasts, which undergo cellular senescence, suggesting
that Sp1 may play a role in the induction of senes-
cence. Although these studies show that p16 is modu-
lated by changes in Sp1 protein levels, they do not
provide convincing evidence demonstrating that up-
regulation of p16 during senescence is Sp1-dependent
[190]. Sp1 levels are also reportedly decreased during
DNA damage-induced cellular senescence. Decreased
Sp1 levels in response to DNA-damaging agents were
dependent on ATM and ATR kinases [317]. Phosphor-
ylation of Sp1 by these kinases is discussed below;
however, phosphorylation of Sp1 by ATM and/or
ATR has not been shown to influence its transcrip-
tional activity or stability [317].
Telomerase
Telomeres are a major determinant of replicative
potential. They protect chromosome ends by prevent-
ing chromosomal fusions, and thereby maintain geno-
mic integrity. In normal cells, telomere length
decreases during DNA replication in a phenomenon
known as the ‘end replication problem’. Once telomere
loss reaches a critical level, a cell enters senescence. In
immortalized cells, telomere length is maintained by a
special reverse transcriptase called telomerase. Most
tumor cells show increased expression of telomerase,
which is suppressed in most differentiated tissues. Sp1
is a key transcriptional regulator of telomerase subun-
its, and, for this reason, is essential in the establish-
ment of replicative immortality in cancer cells.
Initial characterization of the hTERT gene, which
encodes the catalytic subunit of telomerase, revealed
five binding sites for Sp factors and a binding site for
the Myc transcription factor, as well as lack of a
canonical TATA box (Fig. 2) [174,179,180]. Investiga-
tions showed that the five Sp binding sites in the
hTERT promoter supported transcription at a level
> 90% that of the of the full promoter, and E–boxregulation through Myc proteins required the Sp sites
[174].
The gene encoding the RNA component of telomer-
ase (hTERC) also contains multiple Sp binding sites
(Fig. 2) [181,318]. Although this promoter contains a
TATA box, it was found to be non-functional. Sp1
participates in hTERC expression; however, a CCAAT
box that binds the factor Nuclear Transcription Factor
Y Alpha (NF-YA) is sufficient for basal transcrip-
tional activity [318]. Of the four Sp binding sites in the
hTERC promoter, two were found to positively regu-
late hTERC expression, while the other two were
found to repress hTERC expression [317]. The Sp site
with the strongest effect on regulation was the site
closest to the CCAAT box, and this site negatively
regulated promoter activity [318]. Sp1 and NF-YA
have been shown to interact, and this interaction is
reduced by O–GlcNAcylation of Sp1 [125,319].
Increased O–GlcNAcylation is associated with cancer,
suggesting a potential mechanism for Sp1-dependent
increases in hTERC expression due to increased glyco-
lytic flux in tumors.
Sp1 has been shown to be associated with other fac-
tors that positively or negatively regulate telomerase
genes. One study addressed the involvement of MBD1-
containing chromatin-associated factor 1 (MCAF1,
ATF7IP), which binds Sp1 and promotes hTERT and
hTERC expression. It was also shown that decreases
in either MCAF1 or Sp1 decreased binding of RNA
polymerase II and Excision repair cross-complementa-
tion group 3 (ERCC3) to the hTERT and hTERC pro-
moters [320]. Multiple regions of the C–terminus of
Sp1 were found to interact with multiple portions of
MCAF1. The authors of this study suggest that
decreases in either MCAF1 or Sp1 affect the relative
methylation of the various CpG-rich regions of the
hTERT and hTERC promoters, resulting in long-term
suppression of telomerase expression in normal tissue
[320].
Sp1 also regulates hTERT expression by modulating
histone modifications, including acetylation. Even dur-
ing hTERT repression, Sp1 and Sp3 reportedly remain
bound to the promoter, and recruit histone deacetylas-
es [108]. Specifically, the N–terminal portion of Sp1
binds HDAC2 [108], decreasing histone acetylation at
the hTERT promoter and repressing hTERT expres-
sion. Further, studies using HDAC inhibitors also
implicated HDACs in hTERT repression by Sp1 [105].
233FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
K. Beishline and J. Azizkhan-Clifford Sp1 and the hallmarks of cancer
Sp1 probably recruits additional HDACs such as
HDAC1, together with other factors associated with
HDAC complexes.
In addition to acetylation, histone methylation is
important for Sp1-dependent hTERT expression,
although methylation functions upstream of Sp1 bind-
ing. Liu et al. identified SET and MYND domain-con-
taining protein 3 (ZMYND1) as a specific histone
methyltransferase that is important for maintaining
histone H3 lysine 4 trimethylation of the hTERT pro-
moter [175]. In the absence of ZYMND1 and histone
H3 lysine 4 trimethylation, binding of both Sp1 and c-
Myc is attenuated, and hTERT expression is signifi-
cantly decreased [175].
Upstream signaling factors may promote Sp1-depen-
dent modulation of hTERT expression. One study
addressed the effect of survivin, another Sp1-regulated
factor, on hTERT expression [173]. Survivin was ini-
tially characterized as a caspase inhibitor that sup-
pressed apoptosis. Additional functions for survivin in
cellular signaling, including mitosis, have been charac-
terized [321]. During mitosis, survivin has been shown
to signal through Aurora B kinase to promote direct
or indirect phosphorylation of Sp1 and Myc to
increase hTERT expression [173]. CDK1 modulates
this signaling, suggesting Sp1 may be an important tar-
get for cell-cycle regulation by CDKs. Further work is
required to understand why survivin is important for
regulating hTERT expression.
Finally, the tumor-associated viruses Kaposi sar-
coma-associated herpes virus and human papilloma
virus (HPV) have been shown to modulate hTERT
expression in an Sp1-dependent manner [177,322],
supporting a role for Sp1 in hTERT-mediated
immortalization/tumorigenesis. These viruses express
the viral proteins Kaposi sarcoma latency-associated
nuclear antigen and HPV E6 protein, respectively,
that either directly or indirectly modulate the hTERT
promoter activity through Sp1 sites [177,322]. This
supports a role for Sp1 in tumor formation in
latently infected cells in both pathologies associated
with HPV and Kaposi sarcoma.
The hTERT promoter provides an excellent example
of the relationship between Sp1 binding sites and
regions of G–quadruplex DNA. G–quadruplex DNA,
or G4 DNA, refers to DNA that has adopted a type
of non-B form DNA structure, involving unique stack-
ing of nucleic acids in G–rich sequences of DNA and
RNA [323]. These sequences are present throughout
the human genome, and are more concentrated in telo-
meric regions, fragile sites for DNA double- and sin-
gle-strand damage, and transcriptional regulatory
regions of genes. A distinct correlation between the
occurrence of G4 DNA and Sp1 binding has been
established [324,325]. Sequence analysis of the hTERT
promoter suggests that G–quadruplex structures may
form within the Sp1 binding motifs, and it was sug-
gested they may negatively affect Sp1 binding [178].
Interestingly, other in vitro studies and biophysical
data suggest that Sp1 binds G–quadruplex structures
that contain the minimal consensus motif (50-GGGCGG-30), as well as G–quadruplex structures
that do not contain this motif [326]. These unique
DNA structures have been shown to both activate and
inhibit transcription, depending on their location rela-
tive to the transcription start site [323]. In addition to
hTERT, these complexes have been identified in the
regulatory regions of a number of genes encoding key
oncogenic factors, underscoring the importance of
understanding how G4 DNA contributes to transcrip-
tional regulation, as well as to the maintenance of
genomic stability.
Evading growth suppression
In the absence of nutrient and growth factor stimula-
tion, most cell populations are growth-suppressed or
quiescent. In addition, cellular stresses and DNA dam-
age may suppress growth to prevent cells from divid-
ing in the presence of a stressful environment. In
cancer cells, resistance to these stress signals may be
generated, and pathways that normally suppress cell
growth may be re-wired. Sp1 and its family members
regulate a number of cellular stress response factors,
such as cell-cycle regulators and DNA damage
response proteins (Table 4). In many of cases, Sp1 acts
as a basal transcriptional regulator, and has not been
shown to transcriptionally regulate these factors in
response to any specific stimulus. However, there is
evidence that Sp1 may modulate the cellular response
to specific stresses, such as that caused by ionizing
radiation exposure.
p21
The cyclin-dependent kinase inhibitor p21 (Waf1/
Cip1) is essential for cell-cycle arrest in response to
cellular stress signals [327]. Based on this and addi-
tional findings, p21 has been implicated in cancer
formation and progression [328]. The regulation of
p21 by Sp1 was first characterized in leukemic cells
[192]. This study identified two main regions in the
p21 promoter that may be bound by Sp1: nucleo-
tides �129 to �99 and �86 to �57 relative to the
transcriptional start site (Fig. 2). These binding sites
are responsible for activation of p21 transcription in
234 FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
Sp1 and the hallmarks of cancer K. Beishline and J. Azizkhan-Clifford
response to okadaic acid and phorbol ester, both of
which induce differentiation in these cells [192].
These sites were more proximal to the promoter
than the originally identified p53 binding sites that
control activation of p21 in response to cell stress
and DNA damage [329].
Multiple signaling pathways are associated with
Sp1 regulation of the p21 promoter. Early studies
showed that signaling through the progesterone
receptor stimulated Sp1-dependent up-regulation of
the p21 promoter. This hormone signaling probably
occurs in a manner similar to androgen and estrogen
receptor signaling through Sp1. In this case, proges-
terone receptor signaling also required the acetyl
transferase p300, which is probably recruited in an
Sp1-dependent manner [330]. The Sp1-dependent reg-
ulation of p21 has also been associated with the
activities of signal transducer and activator of tran-
scription protein 3 and 6 (STAT3 and STAT6); the
direct interaction of Sp1 with STAT6 may account
for the stimulation of p21 expression in breast can-
cer cells [138,331].
Several years after their initial characterization, Pa-
gliuca et al. studied the regulation of a number of
CDK inhibitors, including p21, by Sp factors [332].
They showed that Sp1 was a strong activator of tran-
scription, while Sp3 appeared to only weakly activate
the minimal proximal promoters of p21 and other
family members. This suggested that there may be
some interplay between Sp1 and Sp3 in regulation of
the CDK inhibitors. A number of reports have dem-
onstrated clear roles for Sp1 and Sp3 in activation
and repression, respectively, of the p21 promoter. Ka-
vurma and Khachigian identified an interesting phe-
nomenon regarding the three most proximal (A–C)Sp binding sites [194]; they showed that, although
mutation of all three of these sites abolishes promoter
activity, these three sites are essential for repression,
presumably by Sp3. Interestingly, mutation of the C
and E sites resulted in super-activation of the pro-
moter through Sp1. These studies are in agreement
with earlier work by Koutsodontis et al., who found
that Sp1 and Sp3 bound the proximal Sp binding
sites (A–E), with the highest affinity being observed
for site C. They showed that activation through these
sites was severely repressed by expression of an Sp1
mutant lacking the D domain, which is required for
multimerization and super-activation [193]. A decrease
in the Sp3 level resulted in an Sp1-dependent increase
in expression of p21. Although the decrease in Sp3
protein levels probably has a number of indirect
effects, Sp3 may inhibit Sp1-dependent activation of
p21, and its loss may facilitate Sp1 binding.
Another potential mechanism for Sp3-dependent
suppression of the p21 promoter was recently
proposed. When the three most proximal Sp sites
are bound by Sp1, they support activation; however,
when these sites are bound by Sp3, there is evidence
of polymerase stalling and inhibition of p21 tran-
script elongation. Given additional evidence implicat-
ing the more distal Sp binding sites as repressor
elements [191], it is likely that Sp3 inhibits the p21
promoter by preventing Sp1-dependent super-activa-
tion of transcription, which may occur through loop-
ing, to allow interaction of Sp1 bound at the
proximal sites with that bound at the distal sites.
Binding of Sp3 to either of these regulatory regions
may inhibit looping and super-activation, as shown
at other promoters [28].
The p21 promoter is reportedly methylated on
CpG islands in cancer cells [333]. This methylation
has been shown to inhibit binding of Sp1 and Sp3,
providing a potential mechanism for suppression of
p21 transcription mediated by inhibition of Sp1
binding at the promoter in these cells. The relation-
ship between DNA methylation and Sp1 is discussed
further below. However, p21 provides an interesting
example of how epigenetic modifications silence
genes that are normally positively regulated by Sp1,
even within the context of Sp1 over-expression in
cancer cells.
ATM
Sp1 has been shown to be associated with transcrip-
tional regulation of ATM, a key regulator in the cellu-
lar response to genotoxic stress, specifically DNA
double-strand breaks [130]. Serum starvation resulted
in decreased Sp1 binding to the ATM promoter, prob-
ably as a result of decreased Sp1 protein levels. Treat-
ment of cells with ionizing radiation, which activates
ATM activity, stimulated the ATM promoter through
Sp1. The same study showed a direct association of
Sp1 with the kinase domain of the ATM protein, as
well as the N–terminal, substrate and chromatin-asso-
ciated domains [130]. This evidence suggests that Sp1
is modulated in response to decreased growth signaling
and genotoxic stress, and that ATM may regulate its
own expression by phosphorylating Sp1. ATM has
been shown to modulate transcription of other genes
through Sp1, in addition to other transcription factors,
indicating that this may be a conserved mechanism of
transcriptional regulation in response to genotoxic
stresses [201]. Finally, cells isolated from a patient with
ataxia telangiectasia show disrupted Sp1 regulation
and abnormal responses to EGF signaling, again
235FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
K. Beishline and J. Azizkhan-Clifford Sp1 and the hallmarks of cancer
suggesting that ATM is involved in several Sp1-regu-
lated processes related to evading growth suppression
[263].
Resisting cell death and immune escape
Genetic mutation and changes in cellular signaling
within a cancer cell produce a high level of intracellu-
lar stress, which, under normal circumstances, would
promote cell death. In addition, many tumor cells
withstand external signals from the immune system as
it attempts to eliminate cancer cells from normal tissue
populations. In order for tumors to develop, cancer
cells must be able to overcome the intrinsic and extrin-
sic signals that normally induce apoptosis. Regulation
of a number of pro- and anti-apoptotic factors by Sp1
has been investigated, including B-cell CLL/lymphoma
2 (Bcl-2) [227], tumor necrosis factor (ligand) super-
family, member 10 (TRAIL) and its receptors tumor
necrosis factor receptor superfamily, member 10a/b
(DR4/5) [163,225,226,228], myeloid cell leukemia 1
(MCL1) [314], X-linked inhibitor of apoptosis, E3
ubiqutin protein ligase (XIAP) [230], cellular FLICE-
like inhibitory protein (FLIP) [334], Fas ligand (FasL)
[219,231,233] and BCL-2 antagonist/killer 1 (BAK)
[335]. Tumor cells become resistant to extrinsic apop-
totic signals from the immune system by decreasing
the expression of the cell-surface TRAIL receptors
DR4/DR5 and the TRAIL-specific inhibitor FLIP
[336,337]. Sp1-dependent regulation of these factors
not only promotes tumor cell resistance to apoptosis
and immune escape, but may also help promote cancer
cell sensitivity to induction of apoptosis by modulation
of these factors [163]. Most notably, the Sp1-depen-
dent regulation of the anti-apoptotic factor survivin
(baculoviral IAP repeat-containing 5a, BIRC5 gene),
which is closely associated with neoplastic resistance to
cell death, has been thoroughly studied in a number of
tumor cell types [338]. Survivin promotes cell survival
by inhibiting both extrinsic and intrinsic apoptotic
pathways, making it essential for promoting cell sur-
vival in tumors and an important factor in avoiding
immune surveillance.
The BIRC5 promoter contains six putative GC/GT
box targets for Sp factors that are positively regulated
by binding of both Sp1 and Sp3 (Fig. 2) [339]; muta-
tion of key Sp1 sites abolishes basal levels of expres-
sion of the BIRC5 gene [223]. Furthermore, over-
expression of survivin in tumors is directly associated
with an increase in Sp1 protein [219,340].
In response to apoptotic stimuli, survivin levels
decrease in an Sp1-dependent manner. Although this
decrease potentially occurs through loss of p300
recruitment, the underlying mechanism has not been
thoroughly addressed [222]. As mentioned above, a
number of studies have suggested that the interaction
between p300 and Sp1 is important for Sp1-mediated
transcriptional changes [69,71,222,228,330,341–343].Doxorubicin treatment has been shown to suppress
expression of the BIRC5 gene. This suppression may
occur through Sp1/p53-dependent recruitment of
DNMT1 to the BIRC5 promoter [100]. Recruitment
of DNMT1 allows further recruitment of the histone
methyltransferase G9a and the histone deacetylase
HDAC1, and modification of the promoter by his-
tone H3 lysine 9 dimethylation, resulting in epige-
netic silencing of BIRC5. Although this study shows
a direct interaction between the N–terminal portion
of Sp1 and DNMT1, Sp1 remains constitutively
bound to the BIRC5 promoter even in the absence
of doxorubicin treatment, and recruitment of
DNMT1 to the BIRC5 promoter is dependent on
p53 recruitment. DNMT1 has also been shown to
interact with p53, consistent with p53-dependent
recruitment of DNMT1; this dependence is further
supported by the finding that decreases in BIRC5
expression in response to doxorubicin did not occur
in a p53�/� cell line [100]. In addition, we have
found that induction of apoptosis by DNA-
damaging agents, including doxorubicin, induces cas-
pase-mediated cleavage of Sp1 at Asp183. Caspase-
mediated cleavage creates two peptides, a 30 kDa
portion containing the N–terminal inhibitory domain
and the majority of the A domain, and a 70 kDa
fragment containing the remainder of the protein,
including the B domain and the DNA-binding
domain (B. Torabi & J. Azizkhan-Clifford, Drexel
University College of Medicine, unpublished results).
This cleavage was shown to be involved in induction
of apoptosis, and cells expressing a mutant of Sp1
that cannot be cleaved (Sp1 D183A) displayed
increased resistance to apoptosis. Further studies are
underway to determine how Sp1 cleavage affects the
transcription of key apoptotic factors.
Signaling through the ERK1/2 pathway may modu-
late BIRC5 expression. ERK activity negatively regu-
lates BIRC5 expression, and inhibition of ERK
stabilizes survivin in cells treated with the drug querce-
tin, which down-regulates BIRC5 expression [220]. It
appears that quercetin may negatively regulate Sp1
binding to the BIRC5 promoter, but the mechanism
underlying this effect is poorly understood. There is
evidence that ERK-dependent phosphorylation of Sp1
may both positively and negatively regulate transcrip-
tion of specific genes [59,60,73,287]. Signaling through
ERK may modulate Sp1-dependent regulation of
236 FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
Sp1 and the hallmarks of cancer K. Beishline and J. Azizkhan-Clifford
BIRC5 expression, but more direct evidence is required
to verify whether this is in fact the case.
YM155, which is currently in clinical trials as a
suppressor of BIRC5, functions by decreasing pro-
moter activity of the BIRC5 gene [344]. The effects
of the drug, which binds to the BIRC5 promoter,
appear to be specific to BIRC5. Studies of this drug
suggested that the down-regulation of BIRC5 expres-
sion by YM155 occurs through its binding to
regions that bind Sp1. YM155 does not affect the
Sp1 protein level, but may function by directly or
indirectly, affecting Sp1 localization and binding to
the BIRC5 promoter [344].
Promoting tumor-associated angiogenesis
To fulfill the nutrient and oxygen requirements of
growing tumors, angiogenic pathways are activated. A
large number of cellular factors are associated with
angiogenic pathways, both in tumors and normal tis-
sue. Sp1 is associated with the regulation of a number
of pro- and anti-angiogenic genes, including, but not
limited to, those encoding vascular endothelial growth
factor (VEGF), thrombospondin 1 (TSP–1), platelet-
derived growth factor (PDGF) and urokinase-type
plasminogen activator (uPA) (Table 4) [167,234–236,249,252–254,257,258,345]. VEGF is one of the
most highly studied factors associated with tumor
angiogenesis [346,347], and it is well established that
Sp1 plays an essential role in VEGF regulation.
Early characterization of the VEGF promoter identi-
fied a number of Sp1 binding sites near the transcrip-
tional start site of the VEGF gene (Fig. 2) [243,250].
Initial reports indicated that these Sp1 sites were essen-
tial for VEGF expression in response to PDGF stimu-
lation [237]. Further, regulation of VEGF by Sp1 was
shown to be modulated by interactions with other fac-
tors [236,239,242,249], as well as through kinase sig-
naling [240,244,247].
Under normal cellular conditions, hormone signal-
ing participates in the regulation of VEGF expression.
Estrogen promotes endometrial growth and implanta-
tion through modulation of VEGF [238,239]. Studies
have now shown that association of ERa with the
VEGF promoter is dependent on the Sp1 binding sites
within that promoter [238,239]. This estrogen-depen-
dent signaling is important within the context of breast
cancer. Estrogen stimulates ERa-dependent up-regula-
tion of VEGF in breast cancer cells, and this up-regu-
lation is dependent on the interaction of ERa with
Sp1/Sp3 [249]. In addition, androgen receptor also
associates with Sp1 at the VEGF promoter, and modu-
lates VEGF expression [236]. These findings implicate
Sp1 in hormone-dependent regulation of VEGF expres-
sion.
Several intracellular signaling cascades are associ-
ated with Sp1-dependent activation of VEGF. Reactive
oxygen species (ROS) are associated with activation of
a number of signaling pathways, including Ras/Raf/
MEK/ERK signaling (MEK, MAP kinase-ERK
kinase), that mediate Sp1-dependent up-regulation of
VEGF [247]. ROS signaling appears to modulate Sp1-
but not Sp3-dependent changes [247]. ERK1/2 directly
phosphorylates Sp1 on Thr453 and Thr739, and these
modifications are important for increasing VEGF pro-
moter activity [59].
Hepatocyte growth factor (HGF) signaling, which
modulates PI3K, ERK1/2 and protein kinase Cf(PKCf) pathways to modulate phosphorylation of
Sp1, also promotes VEGF expression [245]. PI3K func-
tions upstream of protein kinase B (Akt), an essential
signaling molecule in growth and proliferative path-
ways. Early studies showed that PI3K function was
associated with VEGF expression. Expression of active
Akt increases VEGF expression through increased
binding of Sp1 to sites in the VEGF promoter [244].
Increased active Akt is also associated with increased
Sp1 phosphorylation, although it is not known
whether this is direct or occurs through other down-
stream signaling kinases [244]. These studies also
showed that Sp1 depletion had no effect on the induc-
tion of VEGF in response to hypoxia, which stimulates
hypoxia inducible factor 1a to modulate VEGF. This
suggests that there are multiple mechanisms for modu-
lation of VEGF transcription that are both Sp1-depen-
dent and -independent.
Sp1 has been shown to interact with PKCf, whichmodifies its DNA-binding domain [121]. This modifi-
cation directly affects Sp1-dependent regulation of
VEGF, as well as the ability of Sp1 to interact with
Von Hippel–Lindau protein (VHL), a tumor suppres-
sor protein that negatively modulates VEGF expression
[121]. VHL mutations are found in tumors associated
with von Hippel–Lindau disease, as well as in other
tumors. Loss of VHL is associated with Sp1-dependent
up-regulation of VEGF mRNA [242]. It is now under-
stood that VHL and Sp1 interact with one another,
and it has been proposed that VHL may suppress Sp1
activity at the VEGF promoter. However, whether
VHL affects Sp1 binding or Sp1 transactivation of
VEGF has not been established.
Sp1 O–GlcNAcylation has also been suggested to
play a role in the regulation of VEGF [246], and this
supported by our work with retinal cells demonstrating
that VEGF is induced by glucose via a mechanism
requiring Sp1 and its modification by O-linked
237FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
K. Beishline and J. Azizkhan-Clifford Sp1 and the hallmarks of cancer
glycosylation [244,348]. Increased O–GlcNAcylation of
proteins has been linked to the altered metabolic state
in cancer cells [349,350], as well as diabetic pathologies
[351,352]. Multiple reports have shown that O–linkedglycosylation of Sp1 by O–GlcNac transferase results
in modulation of Sp1 stability and transcriptional
activity. These data support a role for O–GlcNAcyla-
tion of Sp1 in the transcriptional modulation of
VEGF, as well as that of other genes encoding factors
involved in tumorigenesis and diabetes-associated
pathologies.
Activation of tumor cell invasion and metastasis
High levels of Sp1 protein have been show to correlate
with cancer cell migration and metastasis in a number
of tumor models and patient samples, including gas-
tric, pancreatic and breast cancers [158,160,167]. In
contrast, one group showed a negative correlation
between invasiveness and levels of Sp1 protein in late-
stage lung cancer [169]. These studies underscore the
importance of Sp1 in the regulation of a number of
pro- and anti-invasive factors, including matrix metal-
loproteinases (MMPs), membrane type 1 matrix metal-
loproteinase (MT1–MMP), the MMP inhibitor
reversion-inducing cysteine-rich protein with Kazal
motifs (RECK), E–cadherin and integrin a5 (Table 3)
[60,90,137,285,286,296,299,353].
Matrix metalloproteinases
MMPs participate in remodeling of the extracellular
matrix to facilitate cell migration and ultimately
metastasis. Sp1 has been implicated in the regulation
of MMPs and MMP-like proteins. An early study sug-
gested that Sp1 was a key regulator in the transcrip-
tion of MMP2 [299]. Brg1, a component of the
mammalian SWI/SNF (SWitch/Sucrose NonFerment-
able) complex that is important for activation of the
MMP2 promoter, is probably recruited to the MMP2
promoter through interaction with Sp1, increasing
expression of MMP2 mRNA and subsequently MMP2
protein [90]. This recruitment was associated with
decreased Sp3 binding to the promoter of the gene
encoding MMP2, which, in this context, may have
been acting as an inhibitor of transactivation. This
study suggested a mechanism by which Sp1 positively
regulates MMP2 expression.
The CDK inhibitor p16 was also identified as a neg-
ative regulator of Sp1-mediated MMP2 expression.
p16 expression prevented cyclin A from interacting
with Sp1, probably resulting in decreased CDK-medi-
ated Sp1 phosphorylation [137]. Studies of cyclin A/
CDK effects on Sp1 activity support a pathway in
which CDK2 phosphorylates Sp1 on Ser59, thereby
enhancing the transcriptional activity of Sp1 [52].
These data suggest that p16 inhibits CDK-dependent
phosphorylation of Sp1, resulting in decreased tran-
scription of Sp1 target genes, including MMP2. In
cancer cells, loss of p16 may mediate increased MMP2
gene expression via increased Sp1 transactivation. In
addition, Sp1 regulates p16 expression, as discussed
below.
The cell-cycle regulator S-phase kinase-associated
protein 2 (SKP2) has also been shown to influence
Sp1-mediated MMP expression. SKP2 is an E3 ubiqu-
itin ligase that is over-expressed in cancers. It has been
shown to modulate the stability of the CDK inhibitor
p27, as well as to promote increased invasion and
migration of cancer cells [284]. Over-expression of
SKP2 is associated with increases in both MMP2 and
MMP9, and expression of both proteases requires the
transcriptional activity of Sp1 [284]. This suggests two
possible mechanisms by which SKP2 promotes Sp1-
dependent transcription. First, SKP2 may directly
affect CDK-dependent phosphorylation of Sp1
through modulation of the p27 CDK inhibitor. In this
scenario, over-expression of SKP2 decreases p27
expression, thereby enhancing CDK-dependent Sp1
phosphorylation and thus MMP expression. Alterna-
tively, SKP2 may interact with Sp1 and directly modu-
late its DNA binding, as previously demonstrated
[117]. Further work is required to assess the relation-
ship between Sp1 and SKP2.
In addition to regulating the expression of secreted
MMP molecules, Sp1 also regulates the expression of
MT1–MMP, which encodes an MMP that contains a
transmembrane domain for membrane localization.
Functionally, MT1–MMP proteins also stimulate
migration and metastasis. Conflicting reports suggest
that Sp1 both positively and negatively regulates
expression of MT1–MMP [286,354]. Gene expression
in the endothelial cells of the microvasculature is mod-
ulated by mechanical/shear stress. Shear stress has
been shown to increase Sp1 phosphorylation in these
cells and to enhance Sp1 binding to the MT1–MMP
promoter [286]. This result conflicted with previous
studies showing that increased MT1–MMP expression
requires displacement of Sp1 from the promoter via
binding of the transcription factor Early growth
response 1 (EGR1) [354]. More recently, studies have
shown that MT1–MMP is highly expressed in invasive
prostate tumor cell lines and is positively regulated by
Sp1 [285], further complicating our understanding of
how Sp1 regulates MT1–MMP expression. Addition-
ally, Sp1-mediated expression of MT1–MMP is posi-
238 FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
Sp1 and the hallmarks of cancer K. Beishline and J. Azizkhan-Clifford
tively regulated by signaling through b-akt murine thy-
moma viral oncogene homolog 1 (AKT), JNK and
ERK pathways [285]. The initial studies on Sp1-medi-
ated MT1–MMP regulation were performed in rat
microvascular cells, while more recent studies have uti-
lized human cancer cells lines; it is possible that the
differences in results are due to differences between
normal and cancer cells, cell type or species. It is cer-
tainly not without precedence that Sp1 may both acti-
vate and inhibit, depending on the cellular context.
RECK
In addition to regulating MMPs directly, Sp1 also
transcriptionally regulates inhibitors of MMPs, such as
RECK. Initial observations suggested that Sp1 nega-
tively regulates RECK [287–289] through ERK-depen-
dent phosphorylation of Sp1 (on Thr453 and Thr739),
which increases HDAC1 recruitment to the RECK
promoter, and inhibits RECK transcription [60]. ERK
activity was enhanced by the activity of the EGFR
receptor family member HER-2/neu [60], which is fre-
quently over-expressed in breast cancer.
As with MT1–MMP, Sp1 appears to both positively
and negatively regulate the RECK gene: a common
theme in Sp1-dependent transcriptional regulation. Sp1
is known to recruit both activators and repressors of
transcription to gene promoters, depending on the cel-
lular context and post-translational modifications pres-
ent on the Sp1 protein. This allows Sp1 to
differentially modulate gene expression depending
upon the cellular environment.
E–cadherin
Sp1 may also regulate anti-migratory factors such as
E–cadherin. E–cadherin is a cell–cell adhesion glyco-
protein that is dysregulated in a number of cancers
[355–357]. Decreases in E–cadherin are associated with
increased cellular proliferation and metastasis. The
potential contribution of Sp1 to regulation of the E–cadherin gene CDH1 has been described previously
[292,294]. More recent studies in a mouse lung cancer
model and in human lung cancer cells suggest that Sp1
over-expression increases expression of the CDH1
gene. This likely explains the down-regulation of Sp1
in late-stage lung cancer cells, which would support
metastasis by decreasing cell–cell adhesion [169]. The
role of Sp1 as a positive regulator of E–cadherin is
supported by the finding that expression of the E6
protein in HPV16 decreases Sp1 binding to and trans-
activation of the CDH1 promoter [291]. Finally, CpG
island methylation may modulate expression of the
CDH1 gene in neoplastic tissue [358]. Sp1 participates
in protecting CpG islands from methylation in normal
tissue [293]. The involvement of Sp1 in CpG island
regulation is discussed below.
Clearly, Sp1 is necessary for regulation of transcrip-
tion of a number of factors that participate in cell
migration and invasion. The conflicting reports proba-
bly represent differences between normal and cancer
cells, as well as differences among cancer models. Sp1
positively and negatively regulates both pro- and anti-
invasive factors. Additionally, the expression and func-
tion of all of these factors are further influenced by a
number of other cellular pathways, ultimately resulting
in a balanced cellular phenotype.
Inflammation
Inflammation is recognized as a critical component of
tumor initiation and progression. Infection, chronic
irritation and inflammation contribute to tumorigene-
sis. The tumor microenvironment is infiltrated with
inflammatory cells that produce a variety of cytotoxic
mediators, such as ROS, serine and cysteine proteases,
MMPs, tumor necrosis factor a, interleukins, interfer-ons and enzymes, all of which contribute to inflamma-
tory proliferation, survival and migration. As
discussed above, immune surveillance and secretion of
inflammatory cytokines increases the inflammatory
environment of the tumor. Increased proliferation and
metabolism also adds to the inflammatory environ-
ment of a tumor. Thus inflammation acts as a tumor
promoter in association with many carcinogens.
The relationship between Sp1 and inflammatory sig-
naling is well established. Use of non-steriodal anti-
inflammatory drugs (NSAIDs) in cancer prevention
and in the treatment of various tumor types has been
proposed. Decreased inflammatory signaling in
response to NSAID treatment has been associated with
decreases in Sp1 protein levels, as well as in Sp1-depen-
dent transcriptional activity [234,251,298,359–361]. One
study addressing the direct effect of NSAIDs on Sp1
transcriptional activity demonstrated inhibition of
ERK activity, which decreased phosphorylation of Sp1
and activation of its downstream effector MMP2 [298].
Additionally, treatment with the NSAIDs tolfenamic
acid and aspirin has been shown to decrease Sp1-
dependent transcription. These associations provide a
direct link between the regulation of Sp1 by inflamma-
tory signaling and the regulation of essential tumor-
promoting factors. An interesting epidemiological
study supported the notion that Sp1 is intimately asso-
ciated with inflammation [362]. Colorectal cancer
patients with a wild-type COX2 promoter benefited
239FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
K. Beishline and J. Azizkhan-Clifford Sp1 and the hallmarks of cancer
from NSAIDs and the associated decreased expression
of cytochrome c oxidase subunit II (COX2), whereas a
G?C mutation in the COX2 promoter, which pre-
vented Sp1 binding, allowed no beneficial effect of
NSAIDs in these patients [362].
Sp1 has also been implicated in inflammatory signal-
ing associated with a number of pro-inflammatory
genes. Activation of osteopontin in hepatitis C infec-
tion, which has been linked to hepatocellular carci-
noma, has been shown to be Sp1-dependent;
osteopontin production is at least in part responsible
for the epithelial/mesenchymal transition in progres-
sion of hepatocellular carcinoma. Mina, an epigenetic
gene regulatory protein known to function in multiple
physiological and pathological contexts, including pul-
monary inflammation, cell proliferation, cancer and
immunity, requires Sp1 for its induction [363]. T-cell-
specific T-box transcription factor (TBET), a regulator
of interferon c in natural killer cells, T cells, B cells
and dendritic cells, that responds to pro-inflammatory
cytokines, is regulated by Sp1 binding to its promoter.
Mithramycin, which blocks Sp1 binding, was shown to
down-regulate TBET expression, thereby implicating
Sp1 in the inflammatory response [364].
Acute inflammation leads to an increase in zinc
transport into the cell, probably to help protect
against ROS produced by surrounding inflammatory
cells. This influx in zinc also promotes cytokine pro-
duction and further cytokine signaling. In contrast to
acute inflammation, chronic inflammation leads to
decreases in intracellular zinc, which may contribute
to apoptotic and senescent signaling, as well as geno-
mic instability associated with chronic inflammation.
Decreased Sp1 protein levels and apoptotic signaling
have been associated with cytoplasmic sequestration of
zinc [360]. Zinc is a co-factor for Sp1 DNA binding,
and affects Sp1 protein levels and transcriptional
activity. Zinc is also essential for maintaining the
proper structure of the Sp1 zinc fingers [365]. The
DNA binding ability of Sp1 and its nuclear localiza-
tion are dependent on the presence of zinc [20,365].
Therefore, modulation of cellular zinc levels directly
affects Sp1 activity. The increase in zinc associated
with acute inflammation may activate Sp1, and may
help promote tumor-promoting events such as tran-
scriptional up-regulation of factors required for sur-
vival. Zinc levels are elevated in tumors, as well as in
the surrounding tumor micro-environment, but remain
low in the circulating blood serum of cancer patients
[366]. Studies addressing how changes in zinc levels in
response to inflammatory signals affect Sp1 activity
may provide interesting insight into how Sp1 promotes
tumor formation.
Sp1 functions in maintaining genomic stability
There is a strong body of evidence supporting the
idea that genomic instability and DNA damage pro-
mote genetic changes leading to oncogenic transfor-
mation. Cells must maintain the ability to repair
DNA lesions as simple as base modifications and
replication mismatches, to more threatening DNA
lesions, such as single- and double-strand breaks in
order to protect the integrity of the genome. Proper
recognition and processing of DNA lesions is essen-
tial for maintaining DNA sequence fidelity, and for
preventing large-scale rearrangements of the genome
itself. In addition, proper chromosome segregation
must be maintained during mitosis, through mecha-
nisms at the centromere and kinetochore, the mitotic
spindle and the centrosomes. Errors in chromosome
segregation result in chromosomal instability or the
loss or gain of whole chromosomes (aneuploidy) dur-
ing mitosis.
Sp1 may participate in whole chromosome stability
in mitotically dividing cells. Sp1 localizes to the cen-
trosome in interphase cells [367]. Sp1 depletion leads
to aneuploidy and the formation of micronuclei,
probably due to an increase in centrosome number
as seen in cells lacking full-length Sp1 [367]. This
phenotype appears to be dependent on the first 183
amino acids of the protein, because a mutant Sp1
lacking these residues is unable to rescue the multi-
centrosome phenotype [367].
Of the eight Sp family members, Sp1 is unique in
that it contains SQ/TQ cluster domains within its two
transactivation domains; SQ/TQ cluster domains are
targets for phosphorylation by PI3K-like kinases,
including ATM. Sp1 is phosphorylated by ATM in
response to a variety of DNA-damaging agents and
viral infection [49,54,55]. This phosphorylation, which
may be visualized as a 10 kDa electrophoretic mobility
shift from 95 to 105 kDa, or detection by an antibody
that recognizes Sp1 phosphorylated on Ser101, occurs
largely on serines and is rapid and sustained for sev-
eral hours after DNA damage. The disappearance of
this phosphorylation is coincident with the disappear-
ance of the phosphorylated form of Histone H2A vari-
ant X (cH2AX), and presumably the completion of
DNA repair. The mobility shift is probably due to
phosphorylation of several serine residues, and phos-
phorylation of Ser101 appears to be a priming site,
based on the complete absence of phosphorylation of
a mutant of Sp1, S101A, in response to ionizing radia-
tion [54]. It is important to note that hyper-phosphory-
lation of Sp1 is also associated with proliferation
signaling [368,369], although the residues that are
240 FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
Sp1 and the hallmarks of cancer K. Beishline and J. Azizkhan-Clifford
phosphorylated during proliferation and damage are
not all known. Importantly, ATM promotes Sp1-
dependent increases in gene transcription even in
serum-starved cells [370].
Cells depleted of Sp1 show increased sensitivity to
DNA damage at low levels of ionizing radiation, and
this sensitivity can be rescued by expression of wild-
type Sp1, but not by expression of the phosphorylation
mutant S101A [54]. This suggests that Sp1 phosphory-
lation is required for its function in response to dam-
age. Sp1 also regulates transcription of a number of
factors that respond to DNA damage. Interestingly,
microarray analysis of cells lacking Sp1 compared to
normal cells, and of cells exposed to ionizing radiation,
both in the presence and absence of Sp1, suggests that,
in response to ionizing radiation, a relatively small
number of genes are up regulated in an Sp1-dependent
manner (K. Beishline & J. Azizkhan-Clifford, Drexel
University College of Medicine, Paules et al., National
Institute of Environmental Health Sciences, unpub-
lished results). Although Sp1 depletion alone is associ-
ated with a large number of gene expression changes,
evidence suggests that the response of Sp1 to genotoxic
stress may not be primarily transcriptional. Most nota-
bly, nuclear lysates isolated from cells after DNA dam-
age showed no increase in Sp1 binding to its cognate
binding site [49]. In addition, unlike a number of other
transcriptional regulators modulated by ATM in
response to DNA damage, Sp1 is recruited to sites of
DNA breaks (ionizing radiation-induced foci), where it
co-localizes with phosphorylated ATM and other
repair factors [74]. The presence of Sp1 at these sites
of damage, an observed decrease in double-strand
break repair in cells depleted of Sp1, and the rescue of
this repair defect by a transcriptionally inert portion of
the protein [74] suggest that Sp1 plays a role in the
repair process, rather than a transcriptional role, in
response to DNA damage-associated stress signals
Sp1, DNA methylation and cancer
Methylation of CpG sequences in DNA was first iden-
tified in microorganisms in the 1960s, and has been an
area of intensive investigation in eukaryotic cells since
the 1980s, when its importance in vertebrate develop-
ment and genetics was recognized [371]. Clusters of
CpG sequences, known as CpG islands, were mapped
to multiple regions throughout the genome, including
the 50 ends of housekeeping genes and a number of tis-
sue-specific genes. After years of research, it has
become apparent that DNA methylation and CpG
islands are essential for the epigenetic regulation of
genes throughout embryonic development, cellular
differentiation, and a number of disease states, includ-
ing cancer [372].
Over 20 years ago, researchers showed that CpG
islands often contained Sp1 binding sites. These
sequences, present in regulatory regions of housekeep-
ing genes and other essential genomic loci, were pro-
tected from hyper-methylation during stages of
embryogenesis, when methylation patterns are gener-
ated [373]. Further analysis of DNA methylation in
mammalian systems suggested that changes in methyl-
ation of DNA act to activate or repress transcription,
supporting a role for Sp1 in protecting the 50 regionsof housekeeping and other genes from being silenced
epigenetically [374]. Furthermore, there is evidence to
suggest that when methylation spreads into Sp1 bind-
ing elements, it inhibits binding of Sp1 and further
contributes to the silencing of transcription [375]. All
of these data suggest a role for Sp1 not only in the
dynamic regulation of gene transcription, but also in
epigenetic regulation through controlling long-term
gene silencing by DNA methylation.
The understanding of how Sp1 and its family mem-
bers contribute to the methylation status of CpG
islands is derived from a large number of studies
addressing how changes in methylation of oncogenes
and tumor suppressors in neoplastic tissues may be
attributed to Sp1. An example is methylation of the
gene RASSF1A, encoding an Sp1-regulated tumor sup-
pressor that is frequently methylated in cancers [376].
In RASSF1A, changes in histone modifications lead to
eviction of Sp1 from the promoter region, and the loss
of Sp1 allows CpG methylation of the promoter. In
proliferating epithelial cells in vitro, de-acetylation of
histones at the RASSF1A promoter and replacement
of the acetylation marks by histone H3 lysine 9 trime-
thylation accompanied decreased transactivation by
Sp1. Cells within the population that were able to
overcome senescent barriers were found to maintain
suppression of RASSF1A gene expression through
CpG methylation of the Sp1 binding sites. The pro-
moters of other factors important in cancer signaling
reportedly have altered methylation states during
tumorigenesis [332,333,377], suggesting that the
increased levels of Sp1 observed in cancer cells may
not overcome the suppression of selective genes
through promoter methylation. It is important to note
that current studies addressing methylation patterns in
senescent cells compared to cancer cells suggest that
only very specific patches of methylation are important
in cancer phenotypes, while general hypomethylation
of the majority of the genome is a common trait in
both senescent and cancer cells [378]. In senescent
cells, these patterns suppress the expression of prolifer-
241FEBS Journal 282 (2015) 224–258 ª 2014 FEBS
K. Beishline and J. Azizkhan-Clifford Sp1 and the hallmarks of cancer
ative genes but protect the continued expression of
necessary housekeeping genes. These patterns suggest
protection of CpG islands within promoter regions but
addition of methylation marks on upstream and down-
stream elements [378]. These events correlate well with
findings that Sp1 protects from methylation CpGs to
which it is bound within promoters, while also sup-
porting studies in which Sp1 was found to promote
CpG methylation of surrounding regions through
interaction with factors such as DNMT1 [100,378].
The literature supports a strong role for Sp1 in deter-
mining the epigenetic status of both normal and cancer
cells, and further studies are required to shed addi-
tional light on this complex regulation.
Concluding remarks
The studies discussed in this review, which focuses on
the role of Sp1 in cancer, demonstrate that Sp1 is a
highly regulated transcription factor that is involved in
regulating expression of a large number of genes that
contribute to the ‘hallmarks of cancer’. Sp1 both acti-
vates and suppresses the expression of a number of
essential oncogenes and tumor suppressors, as well as
genes involved in a number of essential cellular func-
tions, including proliferation, differentiation, the DNA
damage response, apoptosis, senescence and angiogen-
esis. Given the apparently opposing effects of Sp1, for
example in regulating oncogenes and suppressors, pro-
survival genes and pro-apoptotic genes, a more com-
plete mechanistic understanding of its activities on dif-
ferent promoters and under different cellular
conditions is essential before it may be exploited as a
target for cancer treatment. Additionally, efforts are
required to develop inhibitors that inactivate specific
functions of Sp1. Further study of its non-transcrip-
tional functions is also essential for understanding its
full role in tumorigenesis.
Author contributions
Dr. Beishline and Dr. Clifford worked together to
develop the outline and gather the literature.
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