Sp1 and the 'hallmarks of cancer

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: [email protected]

(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.


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.



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



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]



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 – –



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.



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



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].



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



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



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



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



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-



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



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



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-



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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Sp1 and the 'hallmarks of cancer