Current Gene Therapy, 2006, 6, 17-33 17

1566-5232/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd.
Targeting Transcription Factors for Cancer Gene Therapy
Towia A. Libermann and Luiz F. Zerbini*
BIDMC Genomics Center, Beth Israel Deaconess Medical Center and Harvard Medical School, 4 Blackfan Circle,
Boston, Massachusetts 02115, USA
Abstract: A high proportion of oncogenes and tumor suppressor genes encode transcription factors. Deregulated expres-
sion or activation and inactivation of transcription factors as well as mutations and translocations play critical roles in tu-
morigenesis. Furthermore, the majority of oncogenic signaling pathways converge on sets of transcription factors that ul-
timately control gene expression patterns resulting in tumor formation and progression as well as metastasis. Under nor-
mal physiological conditions whole sets of genes with similar functions are regulated by highly specific, tightly regulated
upstream transcriptional regulators, whereas in cancer aberrant activation of these transcription factors leads to deregu-
lated expression of multiple gene sets associated with tumor development and progression. The activity of these transcrip-
tion factors can be modulated by multiple mechanisms including posttranslational modifications. Activation or inactiva-
tion of transcription factors promote cancer development, cell survival and proliferation and induce tumor angiogenesis.
Since many of these transcription factors are inactive under normal physiological conditions and their expression and ac-
tivities are tightly regulated, these transcription factors represent highly desirable and logical points of therapeutical inter-
ference in cancer development and progression. Three major families of transcription factors have emerged as important
players in human cancer and are validated targets in drug discovery for cancer therapy: 1) the NF-κB and AP-1 families of
transcription factors, 2) the STAT family members and 3) the steroids receptors. This review aims to elucidate the diver-
gent molecular mechanisms involved in the deregulated activation of transcription factor signaling in malignant transfor-
mation, although additional transcription factor families such as the Ets factors, ATF family members, basic helix-loop-
helix transcription factors etc. are additional critical transcriptional regulators in human cancer. We explore new ap-
proaches to specifically inhibit these transcription factors in cancer in order to validate them as a drug targets. Efforts to
develop novel viral vectors for therapeutic applications are also discussed.
Keywords: Transcription factors, apoptosis, cancer.
Over the past years, progress made in cancer biology,
genetics, and biotechnology has led to a major change and
readjustment in cancer therapy design and development.
Biological mechanism-based therapy is designed to act on
cellular and molecular targets that are causally involved in
the formation, growth, and progression of human cancers.
Drugs targeting signaling pathways associated with cancer
may have greater selectivity for cancer versus normal cells
and may produce better anti-tumor efficacy and lower host
toxicity. The development and progression of cancer is
thought to be the consequence of multiple genetic and epige-
netic alterations [Strausberg et al., 2004]. These range from
methylations, point mutations, insertions and deletions to
chromosomal translocations and amplifications in the tumor
cell's DNA [Strausberg et al., 2004; Balmain et al.,2003;
Popescuet et al., 2000]. These modifications exert their
pathologic effects by perturbing the function of two broad
categories of gene defects: gain of function resulting in en-
hanced expression or activation of oncogenes, and loss of
function resulting in the repression or inactivation of tumor-
suppressor genes.
*Address correspondence to this author at the Beth Israel Deaconess Medi-
cal Center (BIDMC) Genomics Center and Dana Farber/Harvard Cancer
Center Cancer Proteomics Core, Beth Israel Deaconess Medical Center and
Harvard Medical School, 4 Blackfan Circle, Boston, Massachusetts 02115,
USA; Tel: 617-6670760; Fax: 617-9755299;
Among several novel promising approaches that have
recently entered the scene of anti-cancer therapy, inhibition
and targeting of oncogenes and/or reactivation of tumor-
suppressor genes are of particular interest as potentially
highly synergistic anti-cancer therapies [Futreal et al., 2001;
Green et al., 2002]. Despite the genomic complexity, hetero-
geneity and instability of tumors, a limited number of spe-
cific oncogenes and tumor suppressor genes appear to be
indispensable for the maintenance of tumor growth and pro-
gression [Green et al., 2002]. The consequences of targeting
a given oncogene or tumor suppressor gene are likely to de-
pend upon the particular type of tumor as well as the context
of other genetic lesions within this tumor.
A major portion of oncogenes and tumor suppressor
genes encode transcription factors, which control gene ex-
pression patterns and signaling pathways in cancer [Darnell,
2002]. Aberrant transcription factor activity is the result of
numerous mechanisms such as changes in expression, pro-
tein stability, protein-protein interactions, and posttransla-
tional modifications among others [Blume-J ensen et al.,
2001], leading to deregulation of gene sets involved in pro-
moting cancer cell survival, proliferation and inducing tumor
angiogenesis and metastasis [Blume-J ensen et al., 2001;
Darnell, 2002].
Many transcription factors recruit co-activators and/or
co-repressors to establish a chromatin modification and re-
modeling complex

at the site of target gene promoters and
18 Current Gene Therapy, 2006, Vol. 6, No. 1 Libermann & Zerbini
enhancers [Evan et al. 2001; Ravi et al., 2004]. Disruptionof
the normal function of transcriptional co-activators or co-
repressors is a common step in malignant transformation
[Evan et al., 2001; Young et al., 2003]. Although transcrip-
tion factors have not been the favorite targets in drug devel-
opment, recent efforts have resulted in identification of sev-
eral small molecule drugs that directly or indirectly affect
transcription factor activity. Transcription factors can be
targeted at various levels including the inhibition of their
interactions with co-repressors, co-activators and other inter-
acting proteins (i.e. level-two proteins) or

blocking their
binding to DNA. Protein interactions occur

through the sur-
face contacts between proteins, with affinities in the high
nanomolar to micromolar range. However, the full potential
of transcription factors as novel targets for cancer therapy
has not been realized and many challenges remain, from the
validation of novel targets to the design of specific agents to
the evaluation of these agents in both preclinical and clinical
settings. Such proteins will be the main focus of this article.
We will discuss progress made in the discovery of transcrip-
tion factors and their signal transduction pathways, in the
context of their potential as targets for cancer therapy with
particular emphasis on a few selected transcription factor
One of the major transcriptional circuits implicated in
inflammation is the NF-κB/IκB pathway [Verma, et
al.,1995; Ghosh et al., 1998; de Martin et al., 1999; Grilli et
al.,1999; Karin et al.,2000]. The NF-κB/rel family consists
of at least five members, p50, p65, c-rel, relB, and p52,
which form homo- and hetero-dimers [Ghosh et al.,1998; de
Martin et al., 1999; Grilli et al.,1999] (Fig. 1). In unstimu-
lated cells, NF-κB is retained in the cytoplasm through inter-
action with inhibitory proteins called IκBs. Rapid phos-
phorylation and degradation of IκB in response to pro-
inflammatory cytokines such as IL-1 and TNF-α, endotox-
ins, viruses, and UV, as well as other primarily inflammatory
stimuli, growth factors and trauma leads to release and
translocation of NF-κB to the nucleus where NF-κB binds to
regulatory elements within the promoters or enhancers of
target genes [Ghosh et al.,1998; de Martin et al., 1999; Grilli
et al.,1999] (Fig. 2). NF-κB is involved in the regulation of a
large set of inflammation response genes including cytokines
and chemokines, acute phase proteins, cell adhesion proteins,
inducible nitric oxide synthase (iNOS), immunoglobulins,
and viral genes [Ghosh et al.,1998; de Martin et al., 1999;
Grilli et al.,1999] as well as cell cycle regulatory and anti-
apoptotic genes. Most of these genes are directly regulated
by NF-κB via high affinity binding sites within the respec-
tive promoter regions (Fig. 2). Furthermore, NF-κB interacts
and synergizes with various other transcription factors such
as C/EBP, AP-1, HMG(I), and Sp-1 in transactivating its
target genes [Brickman et al., 1999]. These protein-protein
interactions are tightly regulated, since interruption of a sin-
gle component can almost completely abolish transcriptional
activation as shown for the IL-6 and IL-8 genes [LeClair et
al., 1992]. Knockouts of several members of the NF-κB/rel
family, IκBs or the upstream IKK IκB kinases have estab-
lished the critical, but distinct roles of the different compo-
nents in inflammatory processes and keratinocyte differen-
tiation [Li et al., 1999; Li et al., 2002].
Fig. (1). Mammalian nuclear factor-κB (NF-κB) members. The nuclear factor-κB family comprises five members: p65/RelA, c-Rel, Rel
B, p50/NF-κB1 and p52/NF-κB2. They have a structurally conserved amino-terminal Rel homology domain that contains the dimerization,
nuclear localization and DNA-binding domains. c-Rel, RelB and p65/RelA proteins also have a carboxy-terminal non-homologous transacti-
vation domain.
Targeting Transcription Factors for Cancer Gene Therapy Current Gene Therapy, 2006, Vol. 6, No. 1 19
Apoptosis and Cell Survival
NF-κB has been implicated in cell survival and inhibition
of apoptosis and demonstrated to be activated by che-
motherapeutic agents in cancer cells [Li et al., 1998; Ban-
nerman et al.,2002; Bhat-Nakshatri et al.,2002; Kosmidou et
al.,2002; Seo et al.,2002]. Since activation of NF-κB can
suppress apoptosis in cancer cells, resistance of cancer cells
to chemotherapeutic agents can be at least partially explained
by NF-κB activation [Baldwin et al.,2001]. NF-κB activa-
tion helps cancer cells to escape programmed cell death and
to survive pro-apoptotic stimuli. Constitutive NF-κB activa-
tion has been directly implicated in tumorigenesis, cancer
cell survival, apoptosis, invasion and metastasis of a variety
of human cancers, including prostate cancer, ovarian cancer,
breast cancer, head and neck cancer, multiple myeloma and
renal cell cancer [Collins et al., 2000; Harwood et al., 2000;
Mitsiades et al.,2002]. Since these types of cancer contain
activated NF-κB prior to therapy, they are expected to be
resistant to many chemotherapeutic drugs a priori. Trans-
formed keratinocytes contain constitutively active NF-κB
which is required for maintaining their transformed pheno-
type. Similarly, NF-κB is activated in pancreatic cancer and
HNSCC cell lines [Arlt et al.,2002; Bancroft et al., 2001;
Bancroft et al.,2002].
The analysis of NF-κB–deficient mice led to the identifi-
cation of NF-κB as an apoptosis inhibitor [Beg et al., 1995].
epithelial cells demonstrate enhanced sensitivity to
TNF-α mediated apoptosis and to a variety of DNA-
damaging chemotherapeutic agents [Beg et al., 1996; Liu et
al.,1996]. Additionally, cells in which NF-κB activation is
inhibited by a superrepressor IκB protein exhibit increased
levels of apoptosis following exposure to DNA damage or
Fig. (2). NF-κB activation and signaling pathway. NF-κB activity is triggered by many pathways, including lipopolysaccharide
(LPS)/Toll-like receptor 4 (TLR4), tumor necrosis factor (TNF) family members TNFα/TNF receptor, lymphotoxin β (LTβ)/ LTβ receptor,
CD40 ligand/CD40, B cell activator factor BAFF/BAFF receptor and interleukin 1 (IL1)/IL1 receptor. The binding of the inducers to their
plasma membrane receptors activates an intracellular signaling cascade that involves the interleukin1-receptor associated kinase IRAK, the
NF-κB-inducing kinase NIK and MAP/Erk kinase kinase 3 (MEKK3) and potentially affects the phosphorylation of the inhibitor of κB (IκB)
kinase (IKK). IKK is composed of two catalytic subunits, IKKα and IKKβ, and one regulatory subunit, IKKγ (also called NEMO). The acti-
vation of IKK leads to enhanced phosphorylation of IκBα, followed by its ubiquitination and then degradation by the proteasome, resulting
in the nuclear translocation of NF-κB heterodimers and transcriptional activation of target genes involved in proliferation, apoptosis and
20 Current Gene Therapy, 2006, Vol. 6, No. 1 Libermann & Zerbini
TNF-α. The NF-κB-mediated escape from programmed cell
death is due to activation of numerous triggers, including the
ligand engagement of death receptors such as tumor-necrosis
factor (TNF-α) [Beg AA et al.,1996; Van Antwerp et al.,
1996]. The anti-apoptotic effect of NF-κB is mediated at
least partially by induction of several anti-apoptotic
genes including c-IAP1, c-IAP2, BCL-xL, XIAP and
GADD45β [Baud et al., 2001; Deveraux et al.,1998;
Deveraux et al., 1999; Wang et al.,1998].
c-IAPs inhibit apoptosis induced by both death receptors
and mitochondria-dependent pathways by inhibiting effector
caspases 3 and 7 and activating pro-caspases 6 and 9 [Wang
et al.,1998]. Induction of c-Iap2 by TNF-α or phorbol
myristol acetate (PMA) +ionomycin is completely blocked
in cells expressing IκB, and two functionally relevant κB
sites are found in the cIap2 promoter [Deveraux et al.,1998].
Similarly, overexpression of XIAP inhibits TNF-α–mediated
apoptosis of cells expressing IκB [Deveraux et al., 1999].
Interestingly, XIAP also inhibits activation of c-J un-N-
Terminal Kinase (J NK) [Tang et al., 2001; Lee et al., 1999].
The importance of J NK in TNF-α–induced apoptosis is very
controversial. Originally, J NK activation and inhibition ex-
periments indicated a pro-apoptotic role for J NK in TNF-α
signaling [Verheij et al., 1996]. Nevertheless, detailed analy-
sis of the TNF-α pathway did not support the pro-apoptotic
function of J NK. Other data even provided evidence for an
anti-apoptotic role for J NK. In contrast, J NK activation plays
a role in ultraviolet-induced apoptosis, which also activates
NF-κB [Tournier et al., 2000]. This controversy about J NK
may be related to the different systems and inducers being
used in different studies. NF-κB has been postulated to in-
duce the expression of a J NK inhibitor that contributes to the
anti-apoptotic function of NF-κB such as XIAP which is able
to restore transient J NK activation to TNF-α–treated RelA-/-
cells [Tang et al., 2001]. The only member of the GADD45
family that had been until recently shown to be involved in
the anti-apoptotic effect of NF-κB is GADD45β [De Smaele
et al., 2001].
The GADD45 gene family encodes three related 'growth
arrest- and DNA damage-inducible proteins, GADD45 α, β
and γ [ Hall et al., 1995]. GADD45 are nuclear proteins that
interact with various cell cycle related proteins such as the
G(2) cell cycle-specific kinase, Cdc2, Cdk1, proliferating
cell nuclear antigen (PCNA), and the cell cycle kinase in-
hibitor p21(waf1) [ Hall et al., 1995; Chen et al., 1996].
GADD45 plays a role in the G2-M checkpoint in response to
DNA damage [J in et al., 2000].
Genotoxic stress induction of apoptosis appears to in-
volve GADD45 mediated activation of the stress-responsive
J NK kinase and/or p38 mitogen-activated protein kinase
(MAPK) [Harkin et al., 1999; Mita et al., 2002]. Interest-
ingly, all three GADD45 family members directly interact
with the upstream kinase MTK1/MEKK4 that activates both
p38 and J NK and induce apoptosis. Other studies, however,
come to opposite conclusions. Overexpression of
GADD45β in RelA-/- fibroblasts completely inhibits TNF-
α–induced apoptosis and cell death induced by DNA-
damaging agents as well as prolonged J NK activation in re-
sponse to TNF-α in RelA-/- cells [Fornace et al., 1992]. Re-
cent findings established the GADD45 family as a critical
mediator of apoptosis in cancer cells [Zerbini et al, 2004].
NF-κB mediated cell survival in cancer cells is absolutely
dependent on two GADD45 family members, GADD45α
and γ (Fig. 2). NF-κB-mediated repression of GADD45α
and γ is sufficient for cancer cell survival and repression of
the GADD45α and γ genes is for a large part the result of
NF-κB mediated upregulation of c-myc, another oncogene
and transcription factor frequently overexpressed or translo-
cated in a variety of cancers.
Inhibition of the NF-κB Pathway
Gene therapy approaches to inhibit NF-κB activation,
including overexpression of IκBα by gene delivery, have
been investigated. In immunocompromised mice, inhibition
of NF-κB through adenoviral delivery of a modified form of
IκB sensitized a chemotherapy-resistant fibrosarcoma to
TNF-α induced apoptosis, resulting in tumor regression
[Nakanishi et al., 2005]. In neuroblastoma, inhibition of NF-
κB using an adenovirus encoding the IκBα gene together
with TRAIL expression resulted in apoptotic death in a
greater proportion than TRAIL treatment alone [Karacay et
al., 2004]. In lung cancer, AdIκBα blocked NF-κB activity
in H460 cells and significantly inhibited cell proliferation by
inducing apoptosis. An in vivo study showed the tumor inci-
dence to be significantly lower in mice implanted with H460
cells infected with AdIκBα. Intratumoral injection of
AdIκBα also inhibited tumor growth due to both a blockade
of NF-κB activity and an inhibition of VEGF expression [Ni
et al., 2005]. Furthermore, adenovirus-mediated gene trans-
fer of super-repressor IκBα blocked TNF-induced NF-κB
activation and sensitized oral squamous carcinoma cells to
TNF-induced killing by enhancing TNF-mediated caspase-8
and -3 activation [Chen et al., 2002]. We, furthermore, dem-
onstrated that adenoviral expression of IκBα inhibits pros-
tate cancer formation in SCID mice [Zerbini et al., 2004].
Progress over the last years in cellular and molecular
research has provided new approaches to inhibit target gene
expression using unmodified and modified antisense oli-
godeoxynucleotides (ODN), ribozymes and decoy ODN,
although their practical applications in the clinic are not en-
tirely clear. More recently, the breakthrough technology of
small interfering RNAs has been shown to specifically and
efficiently inhibit target gene expression and to regulate the
transcription of genes in vivo with important therapeutic po-
tential for cancer. We will discuss these approaches in the
next section.
AP-1 (activator protein 1) was one of the first mammal-
ian transcription factors to be identified. However, although
it happened almost 20 years ago [Angel et al., 1987; Lee et
al., 1987], the biological relevance and physiological func-
tions of AP-1 are still being unraveled. AP-1 binding sites
were first identified in the promoter and enhancer elements
of the metallothionein I gene and simian virus 40 (SV40).
The AP-1 transcription factor is a dimeric complex that
comprises various members of the J UN, FOS, ATF (activat-
ing transcription factor) and MAF (musculoaponeurotic fi-
brosarcoma) protein families (Fig. 3). Activity of AP-1 is
regulated through changes in transcription of genes encoding
Targeting Transcription Factors for Cancer Gene Therapy Current Gene Therapy, 2006, Vol. 6, No. 1 21
the divergent AP-1 subunits, control of the stability of their
mRNAs, posttranslational processing such as phosphoryla-
tion and turnover of pre-existing or newly synthesized AP-1
subunits, as well as cell type- and cell state-specific interac-
tions between AP-1 proteins and other transcription factors
and cofactors [Eferl et al., 2003].
A common feature of all these proteins is the evolution-
arily conserved bZIP domain, the collective term for a basic
DNA binding domain combined with a leucine zipper region
[Eferl et al., 2003]. The leucine zipper is responsible for di-
merization, which is a prerequisite for DNA binding medi-
ated by the basic domain. The composition of the leucine
zipper is also responsible for the specificity and the stability
of homo- and heterodimers formed by the various J UN, ATF
and FOS proteins [Wagner, 2001; Eferl et al., 2003].
Whereas the J UN proteins exist as homo- and heterodimers,
the FOS proteins, which cannot homodimerize, form stable
heterodimers with J UN proteins and thereby enhance their
DNA-binding activity (Table 1). Several members of the
ATF family form heterodimers with FOS and J UN family
proteins. The ATF family members bind with high affinity to
the octameric cyclic AMP-responsive element (CRE), which
differs by a single nucleotide from the heptameric AP-1
binding site. Moreover, the ATF family members have dis-
tinct partner specificities. ATF 1 forms only homodimers but
does not combine with other ATF proteins. ATF2, ATFa,
ATF3, ATF4 and ATF6 interact both with themselves and
with specific J UN and FOS family members (Table 1). For
example, c-J un forms stable dimers with ATF2, ATF3 and
ATF4 while c-Fos and Fra1 heterodimerize with ATF4 but
not ATF2 and ATF3 [Chatton et al., 1994]. J UN, FOS and
ATF family members can also associate with certain mem-
bers of the MAF family [Motohashi et al., 1997]. The DNA
binding affinities and transactivation capacities of the AP-1
proteins vary considerably, with significantly different trans-
activation potentials [Ryseck et al., 1991]. Whereas J un, Fos
and FosB are considered strong transactivators, J unB, J unD,
Fra-1 and Fra-2 exhibit only weak transactivation potential.
AP-1 regulates a wide range of cellular processes, including
cell proliferation, death, survival and differentiation (Table
2). The DNA binding of the AP-1 complex to the TRE se-
quence (TPA-responsive element) is rapidly induced by tu-
mor promoters, growth factors, cytokines and oncoproteins
[Eferl et al., 2003].
The functions of the individual AP-1 protein complexes
in normal development have been elucidated using geneti-
cally modified mice [Angel et al., 1991]. These analyses
have revealed that each AP-1 component has specific func-
tions during embryogenesis and organogenesis. AP-1 pro-
teins, such as c-Fos, c-J un, and FosB, can transform cells
efficiently in culture, and have potent transactivation do-
mains. As an example, c-Fos and c-J un, when overexpressed
Fig. (3). The Jun and Fos proteins. The J un and Fos proteins exhibit several domains, including bZIP domain (leucine zipper plus basic
domain), transactivation domains and docking sites for several kinases such as c-J un amino-terminal kinase (J NK), extracellular signal re-
lated kinase Erk, caseine kinase II (CKII) and glycogen synthethase kinase 3β (GSK-3β). These kinases phosphorylate serines and threonine
residues and thereby modulate the activity of both proteins. J NK phosphorylates serine residues within the transactivation domain, regulating
its activity. CKII and GSK-3β phosphorylate threonines 275 and 243 and serine 205 of J un. Erk phosphorylates serines 325 and 331 and
threonine 374 of Fos.
22 Current Gene Therapy, 2006, Vol. 6, No. 1 Libermann & Zerbini
Table 1. Dimerization of the Fos-Jun Family Member
Jun JunB JunD Fos FosB Fra-1 Fra-2
Jun yes yes yes yes yes yes yes
JunB yes yes yes yes yes yes yes
JunD yes yes yes yes yes yes yes
Fos yes yes yes No No No No
Fra-1 yes yes yes No No No No
Fra-2 yes yes yes No No No No
Table 2. Functions of AP-1 Subunits in Various Cellular Processes and Diseases
Gene Name Cellular Process Disease
J unB Differentiation of fibroblasts, keratinocytes,
granulocytes, T cell, bone cells
Stimulates and inhibits proliferation
Promotes angiogenesis
Chronic myeloid leukemia, Hodgkin lymphoma
J unD Differentiation of T cells
Stimulates and inhibits proliferation
Inhibit apoptosis
J un Differentiation of fibroblasts, keratinocytes,
granulocytes, bone cells
Stimulates proliferation
Promotes and inhibits apoptosis
Promotes invasiveness
Squamous cell carcinoma
Fos Differentiation of bone cells
Stimulates proliferation
Promotes and inhibits apoptosis
Promotes invasiveness
Squamous cell carcinoma
Fra 1 Differentiation of bone cells
Promotes Apoptosis
Promotes angiogenesis
Promotes invasiveness
Fos B Stimulates proliferation
∆FosB is involved in Bone cell Differentiation and
stimulation of proliferation
J un proteins preferentially areimplicated in proliferation and apoptosis whereas Fos proteins areinvolved in angiogenesis and tumor invasion. Positive, negativeand even dual func-
tions of individual subunits influencedifferent cellular process, modulating theAP-1 activity and its target genes during malignant transformation.
in mice, induce osteosarcomaand skin and liver tumors, re-
spectively [Wang et al., 1991; Grigoriadis et al., 1993;
Young et al., 1999; Eferl et al., 2003]. In contrast, AP-1
proteins that lack potent transactivation domains, such as
Fra-1, Fra-2, J unB and J unD express weak transforming ac-
tivities [Bergers et al., 1995]. Nevertheless, overexpression
of Fra-1 and Fra-2 can lead to lung tumor formation which
might involve dimerization with other AP-1 proteins that
have an intact transactivation domain [J ochum et al., 2000].
Targeting Transcription Factors for Cancer Gene Therapy Current Gene Therapy, 2006, Vol. 6, No. 1 23
Some AP-1 proteins that lack transforming activity can
actually suppress tumorigenesis [Deng et al., 1993]. The
decision as to whether AP-1 is oncogenic or anti-oncogenic
may depend on the opposing activities of different AP-1
proteins, and is probably influenced by tumor type, tumor
stage and the genetic background. The anti-oncogenic activ-
ity of J unB was confirmed recently using J unB-deficient
mice, and J unB has been shown to express tumor-suppressor
activity in several tissues [Zenz et al., 2003]. J unD appears
to have a negative effect on cell proliferation [Pfarr et al.,
1994]. Mice that lack J unD are viable and do not form tu-
mors spontaneously [Thepot et al., 1994]. The ATF family
members have also been shown to induce transformation.
Although ATF2 overexpression by itself does not induce
transformation, ATF2 potentiates the ability of v-J un to in-
duce proliferation and to significantly inhibit the ability of v-
J un to induce colony formation [van Dam et al., 2001].
Cell Proliferation
A role for AP-1 in the control of cell proliferation has
been suggested based on observations that AP-1 activity is
induced upon mitogenic stimulation and that the various AP-
1 proteins have distinct expression patterns during cell cycle
progression [Angel et al., 1991; Lallemand et al., 1997].
Recent analysis of multiple AP-1 components has demon-
strated that AP-1 proteins have opposite functions in the
regulation of cell proliferation with, sometimes, a marked
anti-proliferative activity. Although rapidly induced in re-
sponse to growth factors, c-Fos, FosB and Fra-1 appear to be
dispensable for cell cycle progression [Gruda et al., 1996].
However, c-Fos/FosB double mutant fibroblasts proliferate
slower, at least partly due to ineffective cyclin D1 induction
in response to serum stimulation. Interestingly, c-J un is a
positive regulator of cell proliferation [Behrens et al., 2000;
Wisdom et al., 1999]. c-J un becomes activated upon phos-
phorylation by J NK kinases and induces, consequently, the
transcription of positive regulators of cell-cycle progression
[Behrens et al., 2000; Behrens et al., 1999]. Divergently,
J unB and J unD are usually negative regulators of cell prolif-
eration. Immortalized J unD-deficient fibroblasts show in-
creased proliferation and are more sensitive to p53-
dependent apoptosis after UV exposure [Weitzman et al.,
2000]. Specific analysis of J unB function in granulocytic
progenitors shows that the loss of J unB expression is directly
responsible for the hyperproliferative phenotype of the
granulocytic progenitors as re-expression of J unB fully re-
verts the hyperproliferative phenotype. [Passegue et al.,
2001]. Accordingly, fibroblasts from transgenic J unB mice
display reduced proliferative capacity [Passegue et al.,
Depending on the particular cellular environment and the
subunit composition and activation status AP-1 can induce
apoptosis or enhance cell survival in specific cell types, in-
cluding human tumor cells [Shaulian et al., 2002]. The effect
of AP-1 activation on cell survival is probably due to differ-
ential regulation of pro-apoptotic and anti-apoptotic target
genes. Certainly, based on the many different forms of AP-1,
their exact functions are likely to be dependent on the spe-
cific composition, posttranslational modifications and pres-
ence of interacting factors [Shaulian et al., 2002]. Activation
of the MAPK cascade can enhance AP-1 activity through the
phosphorylation of distinct substrates. Importantly, the in-
duction of AP-1 by pro-inflammatory cytokines and geno-
toxic stress is mostly mediated by the J NK and p38 MAPK
cascades [Behrens et al., 2000]. Once activated, the J NKs
translocate to the nucleus, phosphorylate c-J un and thereby
enhance its transcriptional activity [Behrens et al., 2000]. As
an example, in neuronal cells, c-J un regulates the expression
of BCL-2 family members that are crucial for neuronal
apoptosis [Whitfield et al., 2001]. In T cells, c-J un and c-Fos
regulate the expression of Fas ligand (FasL), which can trig-
ger apoptosis through the Fas receptor [Kasibhatla et al.,
1998]. J un proteins exert a cytoprotective signal through the
induction of expression of genes that protect cells from cell
death induced by TNF-α [Lamb et al., 2003].
AP-1 also regulates genes that are required for tumor
metastasis. Metastasis and invasive growth of certain epithe-
lial cancers is characterized by epithelial–mesenchymal tran-
sition (EMT), which can be induced by c-Fos and c-J un and
is associated with loss of cell polarity [Lamb et al., 1997].
It is also important to mention that other transcription
factors are regulated by the MAPK signaling cascade. ETS
transcription factors, c-myc, and p53 are examples of tran-
scription factors that play essential roles in the maintenance
of the transformed phenotypes of tumor cells and represent
additional molecular targets for new approaches to cancer
treatment [reviewed by Oikawa et al., 2004 and Darnell et
al., 2002]. Most of them are downstream targets of Ras-
MAPK kinase signaling pathways and their deregulation can
lead to tumor progression, invasion and metastasis.
Inhibiting AP-1
The use of gene therapy methodologies to block AP-1 in
cancer appear to be promising. TAM67, a dominant negative
mutant of c-J un, has been used in different gene therapy ap-
proaches for cancer [Young et al., 1999]. TAM67 has a bZIP
domain, but lacks the transactivation domain and can, there-
fore, still dimerize with other members of the J UN and FOS
families and with other bZIP proteins. As a result, dimers
containing TAM67 are able to bind DNA, but have little or
no transactivation activity [Young et al., 1999]. Furthermore,
these dimers can bind to the p65 subunit of NF-κB and to
other AP-1-interacting proteins, inhibiting NF-κB- as well as
AP-1-mediated activation of genes that are important in hu-
man cancer [Young et al., 2003].
In colon cancer, an adenovirus encoding TAM67 signifi-
cantly inhibited cell proliferation while its transfection into
xenografted HT-29 cell tumors in nude mice significantly
decreased tumor volume on day 21 after treatment. [Suto et
al., 2004]. Interestingly, infection of an adenovirus encoding
A-Fos that inhibits AP-1 DNA binding, induces a significant
decrease in cell viability in drug-resistant human ovarian
cancer cells [Bonovick et al., 2002].
TAM67 and A-Fos dominant-negative mutants have also
been implicated in controlling genes associated with cell
motility and invasion. High levels of both TAM67 and A-
Fos arrest HT-1080 cells in the G1 phase of the cell cycle,
induce changes in colony morphology, and impair direc-
24 Current Gene Therapy, 2006, Vol. 6, No. 1 Libermann & Zerbini
tional motility [Bahassi et al., 2004]. TAM67 also inhibits
breast cancer cell growth, predominantly by inducing in-
hibitors of cyclin-dependent kinases and by reducing the
expression of the cyclins involved in transitioning from G1
into S phase of the cell cycle [Liu et al., 2004]. TAM67 ex-
pression in ras-transformed cells reverses the transformed
phenotype and inhibits tumorigenicity in nude mice
[Kielosto et al., 2004].
STATs were originally discovered as latent cytoplasmic
transcription factors that mediate cellular responses to di-
verse cytokines [Darnell, 1997]. STAT proteins are also ac-
tivated by growth factors via their receptors, such as epider-
mal growth factor (EGF), hepatocyte growth factor (HGF)
and platelet-derived growth factor (PDGF), which all possess
an intrinsic tyrosine kinase activity [Catlett-Falcone et al.,
1999]. Tyrosine phosphorylation of STATs follows upon
binding of cytokines or growth factors to cognate receptors
on the cell surface (Fig. 4). So far, seven STAT family
members have been identified and designated as STAT1,
They elicit divergent biological functions, which include
effects on cell differentiation, proliferation, development,
apoptosis, and inflammation [Akira, 2000; Hirano et al.,
STATs pre-exist within the cytoplasm in an inactive

Tyrosine kinases that mediate STAT activation include
growth factor receptors and cytoplasmic tyrosine kinases
such as J anus kinase (J AK) and Src kinase families. The
J AK family of tyrosine kinases become quickly

activated by
autophosphorylation upon ligand-inducedreceptor activation
[Darnell, 1997; Akira, 2000; Hirano et al., 2000]. Once tyro-
sine phosphorylated, two STAT monomers form dimers
through reciprocal phosphotyrosine-SH2 interactions. Di-
merized STATs rapidly translocateto the nucleus, and bind
to STAT-specific DNA-response elements of target genes to
induce gene transcription (Fig. 4).
STAT proteins act as cytoplasmic signaling proteins and
as nuclear transcription factors that activate a diverse set of
genes, including some that are implicated in malignant de-
velopment [Darnell, 1997; Akira, 2000]. In untransformed
cells, STAT proteins transmit cytoplasmic signals from
polypeptide cytokines and growth factors that have receptors
with intrinsic or associated tyrosine-kinase activity [Darnell,
1997; Starket et al., 1998]. STAT signaling has been shown
to be constitutively activated in a rising number of human
cancers [Buettner et al., 2002]. Constitutive activation of
STAT proteins has been described in a large number of hu-
man cancer cell lines and primary tumors, including blood
malignancies (leukemia, lymphoma, multiple myeloma) and
solid neoplasias (head and neck, brain, breast, lung, pan-
creas, prostate cancers) [Catlett-Falcone et al., 1999; Bow-
man et al., 2000; Coffer et al., 2000; Song and Grandis,
2000; Bromberg, 2002].
Gene knockout studies have defined the biological im-
portance of STAT members in normal cells [Akira, 2000]. In
particular, STAT2 and STAT3 null mice are embryonic le-
thal, consistent with a fundamental role for these STAT pro-
teins in development. Mice with targeted STAT1 gene dis-
ruption are viable, have impaired response to interferons and
show high susceptibility to bacterial and viral infections
[Meraz et al., 1996]. STAT5 knockout mice are viable with
phenotypic defects that are tissue-specific, including defects
in mammary gland development and lactation during preg-
nancy [Liu et al., 1997].
One of the reasons for STATs often becoming persis-
tently activated in cancer is based on the fact that one or
more upstream tyrosine kinases become overactive due to a
variety of genetic or epigenetic alterations. The initial find-
ing that STAT3 is constitutively activated in v-Src trans-
formed cells [Yu et al., 1995] suggested that aberrant STATs
may have key roles in oncogenesis. Recent observations
demonstrated that constitutive STAT3 activation is indeed,
required for oncogenic transformation by v-Src [Levy et al.,
2002]. STAT 5 signaling has also been shown to be impor-
tant during oncogenesis. Constitutive activation of STAT5
accompanies transformation of pre-B lymphocytes by the v-
Abl tyrosine kinase, which is blocked by selective
BCR–ABL kinase inhibitors [Huang et al., 2002]. STAT5 is,
furthermore, persistently activated by the FMS-like tyrosine
Fig. (4). STAT activation pathways. The signal transducers and
activators of transcription (STATs) are activated by many signal-
ing pathways that are frequently activated in cancer cells. Binding
of growth-factor receptors, cytokines and peptides to their recep-
tors results in the activation of intrinsic J AK tyrosine kinases and
non-receptor tyrosine kinases (NRTK) and recruitment of STATs.
The STATs have a SH2 domain and after tyrosine phosphoryla-
tion, dimerize by reciprocal SH2 interactions. Phosphorylated
STATs then translocate to the nucleus where the dimers directly
regulate gene expression. The constant activation of tyrosine
kinases in cancer cells causes constitutive activation of STATs.
Targeting Transcription Factors for Cancer Gene Therapy Current Gene Therapy, 2006, Vol. 6, No. 1 25
kinase 3 (FLT3) receptor in acute myeloid leukemia (AML)
[Levis et al., 2002]. However, in many circumstances, acti-
vated STAT1 seems to act in a pro-apoptotic and anti-
proliferative conduct, showing tumor-suppressor activity that
might antagonize the activities of STAT3 and STAT5 [Levy
et al., 2002].
Significant elevations in the DNA-binding activity of
both STAT3

and STAT5

have been noted in a small sample
of malignantly

transformed breast tissues when compared to
normal tissues [Levy et al., 2002]. In breast tumors, consti-
tutive activation of STAT3 is associated with the induction
of the expression and/or the activity of the EGF receptor
family kinases (HER1/erbB-1, HER2/neu) and of Src, due
mainly to an aberrant expression of EGF-related ligands
and/or their receptors [Buettneret et al., 2002]. Furthermore,
once again in breast carcinoma cells, the coexpression of
synergistically activated c-Src and STAT3 induces the acti-
vation of HGF transcription, which confers increased sur-
vival and growth during progression and metastasis [Hung et
al., 2001].
Cell Proliferation and Apoptosis
STATs regulate several target genes that are important
for cell proliferation and apoptosis of tumor cells. Regulation
of anti-apoptotic members of the BCL-2 family, specifically
, has been associated with STAT3 and STAT5 ac-
tivity in several nonmammary cell lines [Mora et al., 2002].
Inhibition of STAT3 signaling blocked the expression of
in tumor cells and sensitized them to FAS-mediated
apoptosis [Niu et al., 2002]. MCL1 is another anti-apoptotic
gene of the BCL-2 family that is a target of both STAT3 and
STAT5. Blocking either of these STAT proteins in human
tumor cells leads to downregulation of MCL1 expression and
induction of tumor-cell apoptosis [Niu et al., 2002]. Acti-
vated STAT3 also induces the expression of another anti-
apoptotic protein, survivin, which is a member of the in-
hibitor of apoptosis (IAP) family [Buettner et al., 2002].
STAT5 regulates the transcription of cyclins D1/D2 and
of c-myc in some cell types [Bowman et al., 2000; Levy et
al., 2002]. The constitutive activation of STAT5a/b, proba-
bly promotes tumorigenesis by deregulating the cyclin com-
plexes D/CDK4-6, which control progression from the G1 to
the S-phase of the cell cycle [Bowman et al., 2000], and by
deregulation of c-myc-dependent cell growth. The involve-
ment of STAT1 in growth arrest and apoptosis in many cell
types may be explained by its capacity to induce caspase and
p21/waf1 expression [Bromberg et al., 2002; Buettner et al.,
2002; Levy et al., 2002]. These observations make it com-
pelling to examine the role of STAT signaling in malignant
progression to establish whether constitutive STAT activa-
tion present in human tumors is essential for the malignancy.
Inhibiting STATs
STAT3 has been demonstrated to be a potential novel
target for cancer gene therapy. In vitro expression of a
STAT3 variant with dominant-negative properties induced
cell death in murine B16 melanoma cells with no effect on
normal fibroblasts. Significantly, gene therapy by electroin-
jection of the dominant-negative STAT3 expression vector
into preexisting B16 tumors caused inhibition of tumor
growth as well as tumor regression which was associated
with apoptosis in vivo [Niu et al., 1999].
In head and neck cancer, the use of a transcription factor
decoy to selectively abrogate activated STAT3 inhibited
proliferation of cancer cells, but did not affect the prolifera-
tion of normal oral keratinocytes [Leong et al., 2003]. Ac-
cordingly, in vivo liposome-mediated gene therapy with a
STAT3 antisense plasmid efficiently inhibited STAT3 acti-
vation, increased tumor cell apoptosis, and decreased
BCL-xL expression in a head and neck xenograft model
[Grandis et al., 2000]. Furthermore, blockade of STAT5b,
but not STAT5a, using antisense oligonucleotides resulted in
in vivo tumor growth inhibition and abrogation of STAT5
target gene expression in squamous cell carcinoma of the
head and neck (SCCHN) [Xi et al., 2003].
RNA interference has been also utilized to block STAT3
expression. The use of small interfering RNA (siRNA) spe-
cific for STAT3 in prostate cancer cells led to blockade of
STAT3 activation, suppression of prostate cancer growth and
induction of apoptotic cell death [Lee et al., 2004]. The
blockage of STAT3 activation using a transcription factor
decoy approach decreased tumor growth and STAT3 target
gene expression in vivo. In a xenograft model of SCCHN,
daily administration of the STAT3 decoy resulted in de-
creased tumor volumes, abrogation of STAT3 activation, and
decreased expression of STAT3 target genes (VEGF, BCL-
xL, and cyclin D1) compared to treatment with a mutant
control decoy. Blockade of STAT3 with the STAT3 decoy
also induced apoptosis and decreased proliferation, an effect
that was augmented when the STAT3 decoy was combined
with cisplatin, both in vitro and in vivo [Xi et al., 2005].
Steroid hormones play a central role in the development
and progression of prostate and breast cancers [Kumar et al.,
2005]. The biological functions of these and other steroid
hormones are mediated by a family of closely related steroid
hormone receptors (SHRs), with the androgen receptor (AR)
mediating the effects of testosterone and related androgens,
and the classical estrogen receptor mediating the effects of
estradiol [Kumar et al., 2005]. Widely studied are also pro-
gesterone receptors and glucocorticoid receptors. Recent
studies have begun to elucidate the complex pathways
through which SHRs regulate gene expression, and their
interaction with other cellular pathways [Hörlein et al.,
2002]. Conformational changes in SHRs can alter their inter-
actions with transcriptional coactivator and corepressor pro-
teins, resulting in cell type specific responses. SHRs are well
recognized therapeutic targets in cancer [Choi et al., 1999;
Shi et al., 2001]. The identification of multiple proteins and
pathways that mediate the downstream functions of SHRs
provide additional therapeutic targets.
SHRs primarily

regulate the initiation of transcription by
directly binding

to specific DNA sequences in the regulatory
region of target

genes called hormone response elements and
recruiting diverse

additional factors characterized as coregu-
lators along with thebasal transcriptional machinery [Kumar
et al., 2005; Tsai et al., 1994]. For transcription factors to
access DNA the chromatin structure needs to be partially
unwound. Binding of ligand to the SHR results in the release
26 Current Gene Therapy, 2006, Vol. 6, No. 1 Libermann & Zerbini
of corepressor complexes which remodel the nucleosomal
structures, helping the recruitment of the basal transcrip-
tional machinery. Coregulators are generally recruited to

target promoters via protein-protein interactions with the
sequence-specific and context-dependent transcription fac-
tors [Collingwood et al., 1999].
Estrogen receptor (ERs) α and β are members of thester-
oid/thyroid hormone receptor superfamily of ligand-
activatedtranscription factors [Luconi et al., 2002]. Estrogen
receptors contain DNA and ligandbinding domains, which
are critically involved in regulating vascular structures and
functions. Receptor-ligand interactions trigger a cascade of
events, including dissociation from heat

shock proteins, re-
ceptor dimerization, phosphorylation, andthe association of
the hormone-activated receptor with specific regulatory ele-
ments in target genes [Mendelsohn et al., 1999]. ER and
ERß activation may lead to distinct biological

activities even
though they share many functional characteristics. As an
example, the protective effects of estrogens to vascular

in ERß knockout mice are ER dependent [Luconi et al.,
2002; Mendelsohn et al., 1999].
Several growth factors can stimulate ER activity in the
absence of ligand [Ali et al., 2002]. Growth-factor stimula-
tion of ER activity is likely to be mediated by phosphoryla-
tion of the ER. Phosphorylation at several sites increases ER
activity. Phosphorylation of serine 118 is achieved upon E2
binding and is mediated by the cyclin-dependent kinase
CDK7 and the MAPK kinases, resulting in ligand-
independent transactivation [Ali et al., 2002]. Overexpres-
sion of ERBB2 (also known as HER-2 or neu), a member of
the EGF receptor family, also has been shown to be impor-
tant in ER activity. The ERBB2 inhibitor trastuzumab
(Herceptin) [Kurokawa et al., 2000] shows a strong inverse
correlation between ERBB2 expression and ER expression
demonstrating that EGFR inhibitors might prove to be useful
in the treatment of ER-positive tumors that are also EGFR-
or ERBB2-positive. Another signaling pathway that might
have an important role in ER activity is the phosphatidyl-
inositol-3-OH kinase (PI3K)-AKT signaling pathway. ER
phosphorylation by AKT occurs and results in ligand-
independent activation of ER [Campbell et al., 2001].
The androgen receptor (AR), another member of the nu-
clear receptor superfamily of ligand-regulated transcription
factors, is a key regulator of processes involved in the
growth of prostate cancer cells and thus has emerged as a
primary therapeutic target in the management of this disease
[Agoulnik et al., 2003]. The activation of AR from inactive,
chaperone–protein bound state requires the binding of an-
drogens which induces a conformational change in the re-
ceptor structure [Hart, 2002; Schaffer et al., 2004]. This
leads to dissociation of chaperone proteins and receptor di-
merization. In the nucleus, dimerized receptor complex
regulates the transcription of target genes by binding to its
response element in DNA sequences within responsive pro-
moters [Hart, 2002; Schaffer et al., 2004]. The DNA-bound
receptor recruits either coactivators or corepressors to their
target gene promoters. Importantly, receptor conformation is
influenced by the nature of the bound ligand and is a primary
regulator of the cofactor preference of the receptor. Because
coactivator recruitment is an obligate step in nuclear receptor
action and its regulation influences receptor pharmacology,
coactivators have received a great deal of attention as poten-
tial therapeutic targets [Hart, 2002; Schaffer et al., 2004].
Only a few AR mutations have been detected in primary
prostate cancer [Feldman et al., 2001], whereas metastatic
prostate cancer frequently has mutations in the AR [Feldman
et al., 2001]. Mutations also appear to be common in other
crucial AR related pathways [Feldman et al., 2001]. Recent
investigations therefore support the theory that androgen
ablation therapy provides selective pressure to target the an-
drogen signaling pathway [Feldman et al., 2001]. For exam-
ple, therapy with the anti-androgen flutamide might select
for mutant ARs in which flutamide acts as an agonist rather
than an antagonist [Feldman et al., 2001; Taplin et al.,1999].
Even in the TRAMP (transgenic adenocarcinoma of mouse
prostate) mouse model of prostate cancer, in which SV40
large T antigen is overexpressed in the prostate luminal
epithelial cells, mutations in AR frequently develop, and
different types of mutations are found in castrated versus
intact mice [Buchanan et al., 2001]. Increased AR abundance
leads to enhanced ligand-occupied receptor content, even in
the face of reduced androgen concentration. Approximately
30% of tumors that become androgen independent after ab-
lation therapy contain amplifications of the AR gene, result-
ing in increased AR expression, whereas none of the primary
tumors from the same patients before androgen ablation
show AR gene amplification [Feldman et al., 2001].
Although tumors with AR amplification express in-
creased levels of AR, the signal to proliferate presumably
continues to require androgen [Feldman et al., 2001]. It has
been proposed that in the absence of ligand, AR activation
could take place by cross-talk with various growth factor
pathways. For example, it has been demonstrated that epi-
dermal growth factor (EGF), epidermal growth factor re-
ceptor-2 (ERBB2/HER-2), keratinocyte growth factor
(KGF/FGF-7), insulin-like growth factor-1 (IGF-1), protein
kinase A, MAPK as well as IL-6 could activate AR signaling
[Feldman et al., 2001; Kurokawa et al., 2000; Pandini et al.,
2005; Culig, 2004]. Additional mechanisms underlying
ligand independent activation of the AR by these alternative
pathways may involve phosphorylation of either AR or its
associated proteins [Culig, 2004]. One of the most interest-
ing suggested AR-interacting pathways is ERBB2-mediated
signaling. Forced overexpression of ERBB2 in androgen-
dependent prostate cancer cells causes androgen-independent
growth in castrated animals. The ERBB2 gene [HER-2/neu]
encodes a transmembrane glycoprotein that contains tyrosine
kinase activity and belongs to the epidermal growth factor
receptor family [Wen et al., 2000]. The development of an
anti-ERBB2 antibody (trastuzumab) based strategy for
treatment of ERBB2 overexpressing breast carcinomas has
raised the question whether ERBB2 could be a useful target
in the treatment of various types of cancer including prostate
cancer as well [Tubbs et al., 2001].
Inhibiting Steroid Receptors
The discovery of steroid hormones and their nuclear re-
ceptors led to the concept that inhibition of steroid receptor
function by an antagonist prevents tumor growth. While the
first anti-hormones, cyproteroneacetate (CPA) and ta-
Targeting Transcription Factors for Cancer Gene Therapy Current Gene Therapy, 2006, Vol. 6, No. 1 27
moxifen, were found accidentally, deeper understanding of
nuclear receptors as transcription factors enabled more ra-
tional, structure-activity based drug discovery.
One novel strategy for the treatment of advanced prostate
cancer could be the selective inhibition of AR protein ex-
pression by anti-sense oligonucleotides or siRNA molecules.
The controlled induction of gene expression is a powerful
tool for studying aspects of many molecular processes in-
cluding development and oncogenesis. Accordingly, down-
regulation of the human AR caused significant inhibition of
LNCaP prostate cancer growth in vivo [Eder et al., 2003].
The use of oligodeoxynucleotides (ODN) to inhibit human
AR led to AR downregulation which was accompanied by
significant cell growth inhibition and reduced PSA secretion
[Eder et al., 2002; Eder et al., 2003]. Additionally, treatment
with ODNs resulted in significant in vivo tumor growth inhi-
bition of prostate cancer. Retardation of tumor growth was
also significant in castrated mice, whereas the scrambled
control ODN did not exert any effects [Eder et al., 2002;
Eder et al., 2003]. The antisense technology has been also
explored. The inhibition of AR expression in LNCaP pros-
tate tumor cells by using antisense AR ODNs reduced AR
expression with significant cell growth inhibition, strongly
reduced secretion of the androgen-regulated PSA, reduced
EGFR expression, and increased apoptosis [Eder et al.,
2002; Eder et al., 2003].
Similar to the successful approaches described above,
knocking down the AR protein level by a siRNA approach
resulted in significant apoptotic cell death, reduced mito-
chondrial potential and induced caspase-3/6 activation. The
apoptotic response was specifically observed in those
siRNA-transfected cells that harbor a native AR gene [Liao
et al., 2005].
The involvement of ERβ in prostate carcinogenesis has
been hypothesized. Several reports have shown that ERβ
expression is decreased when prostate cells undergo neo-
plastic transformation, suggesting that it could play a tumor-
suppressor role. Adenoviral mediated delivery of ERβ in
prostatic carcinoma cells strongly inhibited the invasiveness
and growth of these cells and induced apoptosis [Cheng et
al., 2004].
ERα mediates breast cancer-promoting effects of estro-
gens and many human breast tumors express both ERα and
ERβ. In this regard, adenovirus-mediated expression of ERβ
changed the phenotype of ERα-positive MCF-7 cells. Estra-
diol increases cell proliferation and causes tumor formation
of MCF-7 cells expressing only ERα. In contrast, introduc-
ing ERβ into MCF-7 cells causes an inhibition of prolifera-
tion in vitro and prevents tumor formation in a mouse
xenograft model in response to estradiol [Paruthiyil et al.,
Finally, among the new approaches to silence estrogen-
regulated genes, the use of fusion proteins to block ER ac-
tivity appears to be promising. Expression of fusion protein
between ERα and the promyelocytic leukemia zinc-finger
(PLZF) protein, a transcriptional repressor that acts through
chromatin remodeling inhibited growth of estrogen regulated
cells when PLZF-ERalpha expression is induced in vitro and
in vivo showing it could be useful for treatment of breast
cancer [Buluwela et al., 2005].
During the last years, progress on using gene transfer
technology to treat cancer has been found useful in target
validation, and several gene therapy approaches have been
evaluated. The two events that have permitted the formula-
tion of the concept of cancer gene therapy are the new under-
standing of the molecular mechanisms underlying oncogene-
sis, and the development of the DNA-delivery vehicles or
vectors. There is now a widespread knowledge base for sev-
eral viral vectors, with unique attributes within each of them
providing versatility and efficiency and potential targets in
cancer cells.
The goal of gene therapy for the treatment of cancer is to
develop specific agents with high toxicity against tumor cells
and minimal pathogenicity to normal tissue. There are three
major frequent themes in cancer gene therapy. The first is the
design of strategies to kill or slow the growth of cancer cells.
The second is the development of vectors and delivery sys-
tems that are safe and efficient. The third major theme is the
translation of preclinical studies into clinical protocols and
trials. Currently, a variety of viral vectors are under investi-
gation as anticancer agents including adenovirus, herpes
simplex virus, reovirus, Newcastle disease virus, and vac-
cinia virus. Several of these vectors have demonstrated proof
of principle in preclinical models and are being tested in
clinical trials [Lotze et al., 2002; Gottesman, 2003].
The viral vector most commonly used in cancer gene
therapy is the adenoviral vector. Adenoviral vectors can be
produced in high titers, free of wild-type virus and have a
broad spectrum of tissue tropism in humans. In addition, it is
also possible to alter its surface fiber proteins to change tis-
sue tropism [Gottesman, 2003]. Retroviruses continue to be
used as possible gene delivery vehicles, although recent
events raise concerns about insertional mutagenesis. Al-
though titers are much lower than for adenovirus, the use of
retrovirus may prove a valuable adjunct allowing local
spread of retroviruses introduced into a tumor [Gottesman,
2003]. Herpes simplex virus is receiving increasing attention
because of its ability to replicate and kill cells very effi-
ciently. Replication competent herpesviruses incapable of
growth in normal human tissues have been shown to produce
cytopathic effects and cell death in cancers. Finally, therapy
with oligonucleotide, strategies that employ antisense RNA,
ribozymes, or siRNA are under development.
RNA interference (RNAi) refers to gene-silencing
mechanisms in which the terminal effector molecule is a
short antisense RNA. RNAi is an evolutionary conserved
post-tanscriptional pathway that was first discovered in the
nematode worm, Caenorhabditis elegans, in 1998 [Fire et
al., 1998]. The original study demonstrated that dsRNA
complementary to a particular gene was more efficient at
silencing the corresponding gene expression than either
strand individually [J orgensen et al., 1996]. RNAi is also a
powerful tool to silence gene expression in a sequence-
specific manner.
28 Current Gene Therapy, 2006, Vol. 6, No. 1 Libermann & Zerbini
Successive studies indicated that silencing correlated
with the processing of dsRNA to small 21-23 nucleotide-
duplex short-interfering RNAs (siRNAs) [Cogoni et al.,
2000] and that a dsRNA-specific endoribonuclease III,
known as Dicer, was responsible for processing dsRNA to
siRNAs [Guta, 2000]. Following ATP-dependent processive
cleavage of long dsRNAs by the enzyme Dicer [Dorsett et
al., 2004], siRNA duplexes with 2-nucleotide overhangs at
the 3' termini are produced. The antisense strand of the
siRNA is then incorporated into an RNA-induced silencing
complex (RISC) and is used to target perfectly complemen-
tary mRNA species. When siRNA lacks this 5' phosphate, it
is rapidly phosphorylated by an endogenous kinase [Dorsett
et al., 2004; Karagiannis et al., 2005], unwound by an ATP-
dependent RNA helicase, leaving the antisense strand ex-
posed to guide RISC to its homologous target mRNA for
endonucleolytic cleavage. The target mRNA is cleaved at a
single site in the center of the duplex region and further de-
graded by endo- and exonucleases [Dorsett et al., 2004;
Karagiannis et al., 2005]. Another gene-silencing mecha-
nism involves a group of small RNA molecules, known as
microRNAs (miRNAs) that are not involved in the pathway
that leads to the production of a protein. They regulate the
expression of mRNAs and correspond to 21- to 22-
nucleotide long, single-stranded RNA molecules. Recent
findings suggest that, if the target mRNA is perfectly com-
plementary, miRNA molecules act in a way similar to siR-
NAs and induce the cleavage of the target mRNA [Hutvag-
ner et al., 2002].
The discovery and characterization of RNAi is providing
us with a new tool for targeted inhibition of gene expression
on many different fronts. A large research effort has been
directed at developing potential short RNA therapeutics for
conditions such as viral infections, cancer and neurodegen-
erative diseases. It is generally accepted that the major ob-
stacle is the inefficient in vivo delivery [Dorsett et al., 2004;
Karagiannis et al., 2005]. Recently, several approaches have
been described for generating loss-of-function phenotypes in
mammalian systems by using RNAi [Kunath et al., 2003].
Attempts to deliver concentrations of siRNAs that are thera-
peutically functional in vivo involve the use of liposomal
carriers and virus-based expression vectors. However, all
these approaches have limited applications and are especially
not applicable for generating a long-term silencing effect in
vivo. Lentiviruses appear to be one of the most promising
viral delivery systems to overcome these limitations in vari-
ous mammalian systems [Buchschacher et al., 2000]. Lenti-
viruses have two key advantages over other gene delivery
systems. First, they can infect non-cycling and post-mitotic
cells. Second and most importantly, transgenes expressed
from lentivirus are not silenced during development and can
be used to generate transgenic mice through infection of em-
bryonic stem cells or embryos [Hanazono et al., 2003; Ikawa
et al., 2003].
Lentivirus vectors can express integrated siRNA effi-
ciently in a wide variety of cell lines and primary cells both
in vitro and in vivo [Tiscornia et al., 2003; Naldini et al.,
2000; Rubinson et al., 2003; Ikawa et al., 2003]. In particu-
lar, genes with embryonic lethal phenotypes can be tested for
function by down-regulating the gene target in any cell type,
location, and developmental time frame. The typical siRNA
lentiviral vector carries a wild-type silencing cassette con-
sisting of a long terminal repeat, a Ψ packing signal, the
central polypurine track (PPT), the U6 promoter followed by
the cytomegalovirus promoter (CMV) and the multiple
cloning site (MCS) and the woodchuck hepatitis virus re-
sponse element (Fig. 5). The MCS is used to clone the DNA
sequences containing siRNA hairpins for the gene of inter-
est. The reverse complement of the sense strand, and five
thymidines are used as an RNA polymerase III transcrip-
tional stop signal. The sequence of the target transcript is
separated by a short spacer [a loop] from the reverse com-
plement of the same sequence (Fig. 5).
Up to now, the most successful strategies using RNA
interference against cancer have been the destructive ones.
The network of signal-transducing pathways combined with
the heterogeneity and chromosomal instability of cancers
makes it difficult to isolate the key genes whose blockage
would irreversibly lead to cancer cell death of all cancer
cells. In most cancers, therefore, it may be necessary to block
pathways at several points, or even to target several path-
ways. The potential use of short RNAs in combination with
conventional therapies such as chemotherapy or radiotherapy
has been explored in cancer [Hannon et al., 2004; Sioud et
al., 2004]. Additionally, there is intense interest in the use of
combinations of apoptosis-inducing agents, for example tu-
mor necrosis factor-related apoptosis-inducing ligand, with
siRNAs that silence genes which inhibit apoptosis such as
BCL-2, FLICE-like inhibitory protein or inhibition of apop-
tosis proteins [Chawla-Sarkar et al., 2004]. Another ap-
proach that has been used to augment the effects of radiation
and chemotherapy in cancer cells is RNAi-mediated silenc-
ing of DNA repair factors [Collis et al., 2003]. Short RNAs
targeting the pivotal proteins, related to DNA damage sig-
naling and repair pathways, have been used to enhance ra-
diation and chemotherapy-mediated cell killing in human
cancer cells [Collis et al., 2003].
RNA interference using a lentivirus small hairpin
(shRNA) delivery system against p65, a subunit of the NF-
κB transcription factor which have well characterized cellu-
lar phenotypes when down-regulated has been reported.
Lowering the levels of p65 led to reduced levels of both
IκBα and IκBβ and also sensitized the cells to tumor necro-
sis factor α-induced apoptosis [Piva et al., 2005]. In another
elegant approach, RNA interference using a lentivirus to
deliver shRNA against STAT3 into mammary tumors in
BALB/c mice led to neither STAT3 protein nor phosphoty-
rosine STAT3 detection and reduced tumor growth [Ling et
al., 2005]. Additionally, it blocked tumor formation of 4T1
cells injected into the mammary fat pad. Invasion activity of
STAT3 knockdown cells was also strongly inhibited. How-
ever, the cell cycle was not affected suggesting the stable
expression of siRNA for STAT3 as a potential therapeutic
strategy for breast cancer [Ling et al., 2005].
Gene therapy is one of the most promising and active
fields in cancer therapy treatment. Although previous gene
therapy protocols have brought some stimulating data, the
poor dissemination of viral vectors has emerged as one of the
major obstacles in cancer gene therapy. In this regard, tar-
Targeting Transcription Factors for Cancer Gene Therapy Current Gene Therapy, 2006, Vol. 6, No. 1 29
geting tumor-specific cells becomes a crucial factor for safe
gene delivery. Viral vectors have been gradually modified in
order to enhance their transduction efficiency and to reduce
their toxicity and immunogenicity. There are potential ad-
vantages of gene therapy over systemic drug treatments: no
resistance has been described against nucleic acid therapeutic
agents in the multiple clinical trials already accomplished; a
single treatment should provide enough therapeutic genes to
carry out the eradication of the tumor, and therefore a sig-
nificant cost reduction is projected. Also, the use of siRNAs
as a lethal weapon against the cancer cell does not predict
many potential side effects.
Proliferating literature of genomics and proteomics stud-
ies have led to the notion that cancer cells have a different
transcriptional pattern compared with normal cells. The
completion of the Human Genome Project andthe potential
for gene-based therapies have raised expectationsthat in the
future, gene-targeted cancer therapy will lead to

efficient and
specific treatments. Numerous transcription factors have
been directly involved in disease pathogenesis and many
more will be in the immediate future. While the lack of 'dru-
gable' transcriptional targets was limiting only ten years ago,
it is now no longer a limiting step in developing appropriate
transcriptional therapeutic strategies. Attempts to target tran-
scription factors using gene therapy approaches have been
carried out by transducing tumor suppressor genes or in-
hibitors of oncogenes. Indeed, clinical trials targeting p53 or
NF-κB are currently performed for several types of cancer.
This review demonstrates that four particular families of
transcription factors have emerged as important players in
human cancer and have been validated as targets for cancer
therapy. Together with the progress obtained in the cancer
gene therapy field, pharmacological therapies are already
being developed to inhibit oncogenic signaling pathways.
Novel clinical treatments such as Bortezomib, Herceptin,
Gleevec, C225 or Iressa, are the leading examples of what
can be achieved with deeper knowledge of tumor cells.
However, a large number of important questions are still
not answered. Inhibition of transcription factors could be
considered a difficult process mainly because protein-protein
interactions involve bigger binding surfaces. The concepts
about the transcriptional repression machinery explain many
aspects of tumorigenesis andprovide targets for specific gene
therapies. Although transcription factor protein and DNA
interactions remainan attractive target for therapeutic inter-
vention, they are also involved in normal cellular physiology
and it is possible that potential

off-target effects might also
mediate clinical response, as transcription alone does not
completely explain for all the abnormalities of

the cancer
cell. A major concern about utilizing transcription factors as
therapeutic targets in cancer is the issue of selectivity. The
global inhibition of a specific transcription factor might re-
sult in serious side effects. Any successful transcription fac-
tor inhibitor used for cancer therapy should avoid, for exam-
ple, suppression of the immune system, which is crucial for
cancer surveillance and treatment. The identification of tran-
scription factor target genes in different types of normal cells
and their transformed derivatives is another important area of
research to understand in more detail the precise function of
transcription factors. By selectively targeting a specific indi-
vidual key-signaling component involved in a particular dis-
ease, it might be possible to minimize systemic toxicity.
Furthermore, approaches that specifically disrupt post-
translational modifications of transcription factors and there-
Fig. (5). Schematic representation of lentiviral vector expressing short hairpin RNA (shRNAs). The siRNA lentiviral vector carries the
shRNA expression cassette, consisting of long terminal repeat, Ψ packing signal, the central polypurine track (PPT) and the U6 pol III pro-
moter, followed by the cytomegalovirus promoter (CMV) and the multiple cloning site (MCS) and the woodchuck hepatitis virus response
element. The MCS is used to clone the sense and antisense sequence of the shRNA of the target transcript separated by a short spacer (a
loop). The putative shRNA structure is shown at the bottom.
30 Current Gene Therapy, 2006, Vol. 6, No. 1 Libermann & Zerbini
fore inhibit only subsets of target genes seem to be another
attractive possibility. There areseveral caveats to consider
such as the best way to inhibit a transcription factor and/or
the necessity of a combination of

more than one agent, or the
use of a more complex approach to

achieve a therapeutic
effect as transcription factors often recruit multiple level-
two/co-repressor/co-activator proteins. In addition, tran-
scription factors may have both opposite and overlapping
functions in cellular proliferation and cell fate. By applying
our current knowledge on in vivo concepts and through the
genetic analysis knockout animals, we will be able to deter-
mine the potential redundant or overlapping roles among
transcription factors.
Finally, even if a specific blockage of a particular tran-
scription factor cannot be achieved, disrupting the function
of a group of transcription factors still offers sufficient op-
portunity for extensive cancer therapeutic searches. In an era
of sophisticated technology accessibility, the potential of
selectively inhibiting transcription factor interactions re-
mains promising.
Supported by NIH grants 1RO1 CA85467, P50
CA090381, P50 CA105009 (T.A.L.).
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Received: J uly 08, 2005 Revised: August 17, 2005 Accepted: August 19, 2005