NF-kappaB and epithelial to mesenchymal transition of cancer.
ABSTRACT During progression of an in situ to an invasive cancer, epithelial cells lose expression of proteins that promote cell-cell contact, and acquire mesenchymal markers, which promote cell migration and invasion. These events bear extensive similarities to the process of epithelial to mesenchymal transition (EMT), which has been recognized for several decades as critical feature of embryogenesis. The NF-kappaB family of transcription factors plays pivotal roles in both promoting and maintaining an invasive phenotype. After briefly describing the NF-kappaB family and its role in cancer, in this review we will first describe studies elucidating the functions of NF-kappaB in transcription of master regulator genes that repress an epithelial phenotype. In the second half, we discuss the roles of NF-kappaB in control of mesenchymal genes critical for promoting and maintaining an invasive phenotype. Overall, NF-kappaB is identified as a key target in prevention and in the treatment of invasive carcinomas.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: Metastasis accounts for the most deaths in patients with hepatocellular carcinoma (HCC). Receptor activator of nuclear factor kappa B ligand (RANKL) is associated with cancer metastasis, while its role in HCC remains largely unknown.PLoS ONE 09/2014; 9(9):e108507. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The impact of cellular oxidative stress in promoting the epithelial-mesenchymal transition (EMT) has been noticed. Our previous study shows that SENP3, a redox-sensitive SUMO2/3-specific protease, accumulates in a variety of cancers, but whether SENP3 and SUMOylation involve in the regulation of EMT is unclear. The present study uncovers a novel role of SENP3 in promoting the EMT process in gastric cancer via regulating an EMT-inducing transcription factor, forkhead box C2 (FOXC2). We demonstrate that the expression of mesenchymal marker genes and cell migration ability are enhanced in SENP3-overexpressing gastric cancer cells and attenuated in SENP3-knockdown cells. A nude mouse model and a set of patient's specimens suggest the correlation between SENP3 and gastric cancer metastasis. Biochemical assays identify FOXC2 as a substrate of SENP3. Meanwhile N-cadherin is verified as a target gene of FOXC2, which is transcriptionally activated by a SUMO-less FOXC2. Additionally, reactive oxygen species-induced de-SUMOylation of FOXC2 can be blocked by silencing endogenous SENP3. In conclusion, SENP3, which is increased in gastric cancer cells, potentiates the transcriptional activity of FOXC2 through de-SUMOylation, in favor of the induction of specific mesenchymal gene expression in gastric cancer metastasis.Oncotarget 08/2014; 5(16):7093-7104. · 6.63 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Breast cancer is one of the most common malignancies of all cancers in women worldwide. Many difficulties reside in the prediction of tumor metastatic progression because of the lack of sufficiently and reliable predictive biological markers and are a permanent preoccupation for clinicians. Manganese superoxide dismutase (MnSOD) may represent a rational candidate as a predictive biomarker of breast tumor metastatic progression, because its gene expression is profoundly altered between early and advanced breast cancer, in contrast to expression in the normal mammary gland. In this review, we report the characterization of some gene polymorphisms and molecular mechanisms of SOD2 gene regulation which allows a better understanding of how MnSOD is decreased in early breast cancer and increased in advanced breast cancer. Several studies display the biological significance of MnSOD level in proliferation as well as in invasive and angiogenic abilities of breast tumor cells by controlling superoxide anion radical (O2-) and hydrogen peroxide (H2O2). Particularly, they report how these ROS may activate some signalling pathways involved in breast tumor growth. Emerging understanding of these findings provides an interesting framework for guiding translational research and suggests to define precisely the clinical interest of MnSOD, as prognostic and/or predicting marker in breast cancer, by associating with some regulators involved in SOD2 gene regulation and other well known biomarkers, in addition to the typical clinical parameters.Free Radical Biology and Medicine 12/2014; · 5.71 Impact Factor
Journal of Cellular Biochemistry 104:733–744 (2008)
NF-kB and Epithelial to Mesenchymal Transition
Chengyin Min,1,2Sean F. Eddy,1,2David H. Sherr,2,3and Gail E. Sonenshein1,2*
1Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts
2Women’s Health Interdisciplinary Research Center, Boston Medical Center, 715 Albany Street,
Boston, Massachusetts 02118-2394
3Department of Environmental Health, Boston University School of Public Health, Boston, Massachusetts
bear extensive similarities to the process of epithelial to mesenchymal transition (EMT), which has been recognized for
several decades as critical feature of embryogenesis. The NF-kB family of transcription factors plays pivotal roles in both
promotingandmaintaininganinvasivephenotype.After brieflydescribingthe NF-kB family anditsroleincancer, inthis
review we will first describe studies elucidating the functions of NF-kB in transcription of master regulator genes that
treatment of invasive carcinomas. J. Cell. Biochem. 104: 733–744, 2008.
During progression of an in situ to an invasive cancer, epithelial cells lose expression of proteins that
? 2008 Wiley-Liss, Inc.
Key words: Snail; Slug; ZEB; Twist; vimentin; MMP-2; MMP-9
Epithelial to mesenchymal transition (EMT),
which has been recognized for several decades
as critical for embryogenesis [Korsching et al.,
2005], has recently been shown to also be
relevant to cancer progression. During EMT of
in situ cancer cells, expression of proteins that
promote cell–cell contact such as E-cadherin
and g-catenin can be lost, and mesenchymal
markers such as vimentin, fibronectin, N-
cadherin and the metalloproteinases MMP-2
and MMP-9 can be acquired, resulting in
[Kang and Massague, 2004]. During embryonic
development in Xenopus, Drosophila and C.
elegans, EMT is regulated by a family of zinc
finger master regulatory proteins, including
Snail, Slug, Twist, ZEB1 and ZEB2/SIP1. In
mammalian cells, this family has recently been
implicated in repression of genes that promote
an epithelial phenotype, thereby inducing tran-
sition to a mesenchymal phenotype.
Several studies have indicated that activity
of the NF-kB transcription factor family is
required for maintenance of an invasive pheno-
Wirth and coworkers identified NF-kB as a
central mediator of EMT in a mouse model
of breast cancer progression [Huber et al.,
2004]. Specifically, inhibition of NF-kB in Ras-
transformed epithelial cells (EpRas cells) led
to a 10-fold reduction in metastases to the
lungs following tail vein injection into nude
mice and to a 3-fold decrease in tumor weight in
a mammary fat pad model. Our group demon-
strated that inhibition of NF-kB activity
reduced the invasive phenotype of 7,12-dime-
thylbenz(a)anthracene (DMBA) carcinogen-
transformed mammary tumor cells driven by
the NF-kB c-Rel subunit [Shin et al., 2006].
More recently, we have shown that the NF-kB
? 2008 Wiley-Liss, Inc.
Chengyin Min and Sean F. Eddy contributed equally to this
Grant sponsor: NIH; Grant numbers: PO1-ES11624, RO1
CA82742; Grant sponsor: Department of Army; Grant
number: DAMD 05-1-0286; Grant sponsor: American
Institute for Cancer Research; Grant sponsor: Avon
*Correspondence to: Gail E. Sonenshein, PhD, Department
of Biochemistry, Boston University School of Medicine,
715 Albany Street, Boston, MA 02118-2394.
Received 10 December 2007; Accepted 14 December 2007
subunit RelB promotes the more invasive
phenotype of estrogen receptor (ER)a negative
or low breast cancers [Wang et al., 2007].
In this review, we first briefly introduce the
of NF-kB in the control of master regulators of
EMT. Then we describe studies elucidating the
functions of NF-kB in transcription of mesen-
chymal genes encoding vimentin, MMP-2 and
MMP-9, critical for promoting and maintaining
a mesenchymal phenotype.
NF-kB FAMILY OF TRANSCRIPTION
FACTORS AND CANCER
NF-kB is a structurally conserved family of
dimeric transcription factors distinguished by
the presence of an N-terminal 300 amino acid
which contains sequences mediating dimeriza-
tion, DNA binding, nuclear localization, and
interaction with inhibitory IkB proteins [Ghosh
and Karin, 2002]. Mammals have five NF-kB
members. The c-Rel, RelB, and p65 (also known
as RelA) subunits are synthesized as mature
products and contain a C-terminal transactiva-
tion domain. The p50 (NF-kB1) and p52
(NF-kB2) subunits are synthesized as longer
precursors, p105 and p100, respectively, that
require C-terminal proteolytic processing. Acti-
vation of the IKKa kinase has been implicated
in processing of p100 and p105 to p52 and p50,
respectively [Senftleben et al., 2001]. Although,
p50 and p52 lack a C-terminal transactivation
with Bcl-3 protein [Bours et al., 1993; West-
erheide et al., 2001; Romieu-Mourez et al.,
2003]. NF-kB factors bind DNA target sites as
hetero- or homo-dimers and have different
activities depending on subunit composition
[Ballard et al., 1992; McDonnell et al., 1992;
La Rosa etal., 1994; Lee et al.,1995; Baek et al.,
2002; Jiang et al., 2002; Hoffmann et al., 2003;
Bonizzi et al., 2004]. NF-kB controls genes
encoding proteins involved in cell growth (e.g.,
Cyclin D1, c-Myc, Gro), adhesion (e.g., VCAM,
ICAM), cell survival (e.g., Bcl-xL, Bcl-2, and
responses (e.g., IL-2, IL-6, IL-8).
expressed, constitutive functional nuclear NF-
kB activity occurs normally only in mature B-
lymphocytes and a few other cell types. In most
cells, inactive NF-kB proteins are sequestered
in the cytoplasm in a complex with an inhibitor
protein, termed IkB. NF-kB can be transiently
activated to enter the nucleus by a variety of
signals. In most cases, activation of NF-kB
proceeds by activation of an IkB kinase (IKK)
its degradation. Two major pathways of post-
translational activation of the NF-kB/IkB com-
plexes have been established: the canonical and
alternative pathways, leading to activation of
p50/RelA or p50/c-Rel and p52/RelB or p50/
RelB, respectively. More recently, we have
described a transcriptional pathway mediated
by p50/RelA or p50/c-Rel and AP-1 complexes,
including c-Jun/Fra-2, which leads to de novo
synthesis of the RelB protein [Mineva et al.,
2007; Wang et al., 2007].
Almost a decade ago, high levels of nuclear
NF-kB were unexpectedly found in human
mammary epithelial cells, and the majority of
primary human and rodent breast tumor tissue
samples [Nakshatri et al., 1997; Sovak et al.,
1997]. It is now well established that primary
human breast cancer tissues constitutively
express functional c-Rel, RelA, RelB, p50, p52,
Wang et al., 2007], as well as the IKKa or IKKb
kinases of the IKK NF-kB activating complex
[Romieu-Mourez et al., 2001]. In addition, we
have shown that the IKK-related kinase IKKe/i
kBactivity and invasivephenotype [Eddy etal.,
been described in some breast cancers [Boehm
et al., 2007]. Moreover, ectopic expression of
tumorigenesis in a transgenic mouse model,
pointing to a potential causal role for aberrant
NF-kB expression in human breast cancers
[Romieu-Mourez et al., 2003]. ERa negative
and low cancer cells in culture display high
nuclear levels of NF-kB binding activity due, in
part, to the lack of ERa-mediated inhibition of
NF-kB [Nakshatri et al., 1997]. ERa has also
been shown to inhibit de novo RelB synthesis
and c-Rel binding and activity in breast
[Belguise and Sonenshein, 2007; Wang et al.,
and Gilmore, 2005]. Aberrant expression of one
or more NF-kB subunit has now been reported
in many solid cancers, including head and
neck, prostate, lung and pancreatic adeno-
carcinomas, and hematopoietic malignancies
734Min et al.
[reviewed in Rayet and Gelinas, 1999]. NF-kB
has been shown to promote the transformed
phenotype and survival of cancer cells, and
these topics have been the subjects of several
recent reviews [Gilmore et al., 2002; Gilmore,
2003; Basseres and Baldwin, 2006; Van Waes,
NF-kB IN REPRESSION OF AN
Several developmentally important tran-
scription factors that induce EMT have been
shown to repress epithelial gene expression. In
the best-studied case, E-cadherin gene trans-
cription is inhibited through specific E-boxes in
the proximal promoter [Nieto, 2002; Peinado
are the Snail-related zinc-finger transcrip-
tion factors Snail and Slug [Nieto, 2002]. Two
dEF1) and ZEB2 (also known as ZFXH1B and
SMAD interacting protein 1 (SIP1)) have also
emerged as important factors for the regulation
of E-cadherin and EMT [Comijn et al., 2001;
Eger et al., 2005]. The findings of Yang et al.
 added Twist, a basic helix–loop–helix
(bHLH) transcription factor, to the growing list
of developmental genes with a key role in
repression of E-cadherin and induction of EMT
[Kang and Massague, 2004; Yang et al., 2004].
Multiple lines of evidence indicate that these
factors are regulated either directly or indi-
rectly by NF-kB.
regulate various aspects of EMT during embry-
progression [Nieto, 2002]. During development
ingression of the early mesodermal cells at
gastrulation and in the delamination of the
neural crest from the neural tube [Nieto, 2002].
Snail mutant mice die at gastrulation due to
defective EMT, in part caused by persistent
E-cadherin expression [Carver et al., 2001]. In
mammalian cells, Snail has been shown to be a
direct repressor of E-cadherin (CDH1) gene
transcription and Snail expression induces a
full EMT and increases migration/invasion in
different physiological and pathological situa-
tions [Batlle et al., 2000; Cano et al., 2000;
the expression of additional epithelial genes,
[Guaita et al., 2002; Ikenouchi et al., 2003;
Ohkubo and Ozawa, 2004; Martinez-Estrada
that have been associated with a mesenchymal
and invasive phenotype, including fibronectin
and metalloproteinase (MMP)-9 [Cano et al.,
2000; Guaita et al., 2002; Jorda et al., 2005].
Snail expression has been detected in different
invasive carcinoma and melanoma cell lines
and, importantly, in invasive regions of squ-
amous cell carcinomas and dedifferentiated
ductal breast carcinomas and hepatocarcino-
mas [reviewed in Nieto, 2002; Peinado et al.,
2004c], supporting a key role for Snail in
induction of EMT and tumor invasion.
Snail activity is regulated at multiple levels.
For example, selective phosphorylation by
GSK3 leads to export of Snail from the nucleus
and to its destruction via the ubiquitin-protea-
some pathway [Zhou et al., 2004]. Mercurio and
coworkers showed that GSK3 inhibition stim-
ulates the transcription of the human gene
encoding Snail (SNAI1) via NF-kB signaling
[Bachelder et al., 2005]. In Drosophila, Snai1
transcription is directly activated by the NF-kB
 localized a region between ?194 and
?78 bp in the human SNAI1 promoter, which is
required for stimulation of SNAI1 expression
by ectopic co-expression of NF-kB p65 [Barbera
et al., 2004]. More recently, NF-kB was identi-
fied as the upstream regulator of Snail expres-
MCF10A cells overexpressing a constitutively
(IGF-IR) [Kim et al., 2007]. Specifically, the
induction of SNAI1 mRNA levels during EMT
can be reversed by inhibition of NF-kB signal-
ing. Together, these observations support an
important role of NF-kB in regulation of Snai1
Slug (Snail2) is a zinc-finger transcription
factor in the Snail superfamily [Barrallo-
has been implicated in the control of gastrula-
tion in invertebrates, and in emergence of
the neural crest. Slug is also required for EMT
ofatrioventricular canal endothelial cells
NF-kB and Epithelial Mesenchymal Transition 735
[Romano and Runyan, 1999] and for mesoderm
formation in the chick [Nieto et al., 1994]. Slug
can also repress endogenous E-cadherin gene
expression [Bolos et al., 2003], and cause
1997]. Slug also downregulates the expression
of the epithelial claudins and occludins genes
[Kajita et al., 2004]. In breast cancer cell
lines, Slug levels were shown to correlate with
loss of E-cadherin transcripts [Hajra et al.,
2002], and with decreased expression of cyto-
keratins 8 and 19 [Tripathi et al., 2005].
Furthermore, elevated Slug expression is a
marker of poor prognosis of breast [Martin
et al., 2005], lung [Shih et al., 2005], colorectal
[Shioiri et al., 2006] and pancreatic cancers
[Hotz et al., 2007].
A recent article identified the Slug gene as a
target of dioxin-activated aryl hydrocarbon
receptor (AhR) [Ikuta and Kawajiri, 2006].
Historically, the AhR has been studied for its
responsiveness to a variety of environmental
chemicals (e.g., polycyclic aromatic hydrocar-
bons, PCBs, and dioxins) and its ability to
transactivate genes encoding phase I cyto-
chrome P-450 enzymes that metabolize endog-
enous substrates (e.g., 17b-estradiol) and some
environmental chemicals into mutagenic inter-
2003]. However, more recent studies have
indicated roles for the AhR, even in the absence
regulation of genes critical for growth [Ma and
Whitlock, 1996; Elizondo et al., 2000; Tohkin
et al.,2000; Abdelrahim etal.,2003; Patel etal.,
2006], apoptosis [Elizondo et al., 2000; Patel
et al., 2006; Wu et al., 2007], and in EMT and
tumor progression [Mulero-Navarro et al.,
2005]. For example, immortalized mouse mam-
mary fibroblasts lacking AhR have impaired
tumorigenicity in a subcutaneous mouse xeno-
graph model [Mulero-Navarro et al., 2005], and
DMBA treatment induced a more highly inva-
cell line [Shin et al., 2006]. Of note, we have
recently shown that ectopic co-expression of
c-Rel and the protein kinase CK2 in mammary
epithelial cells induces AhR levels and Slug
expression, which promote an invasive pheno-
type [Belguise et al., 2007]. Consistently, the
mammary tumor cells from bitransgenic mice
with enforced expression of c-Rel and CK2
displayed elevated levels of AhR and Slug and
a highly invasive phenotype. Thus, NF-kB may
indirectly regulate expression of Slug via the
AhR, even in the absence of carcinogens.
ZEB1 and ZEB2
Members of the ZEB family, ZEB1 and ZEB2/
SIP1, are important mediators of EMT [Comijn
et al., 2001; Eger et al., 2005] that have been
implicated in several different types of tumors
et al., 2005]. ZEB2/SIP1 knock-out mice display
resulting in the loss of migratory behavior of
these cells [Van de Putte et al., 2003]. Further-
more, a role for upregulation of ZEB-family
member during EMT was also demonstrated
during tumor progression [Jechlinger et al.,
2003]. Enhanced ZEB2 expression has to date
been reported in a distinct set of cancers,
including gastric, hepatocellular, ovarian and
breast carcinomas [Rosivatz et al., 2002;
Miyoshi et al., 2004; Elloul et al., 2005]. ZEB2
coordinately represses the transcription of
epithelial cell junctional genes via direct inter-
action with ZEB2-binding sites within the
promoter [Vandewalle et al., 2005]. ZEB1 has
also been found to be upregulated during EMT,
and its ectopic expression is sufficient to induce
the downregulation of E-cadherin and ZO-1,
disintegration of cell–cell junctions, and induc-
tion of mesenchymal marker proteins in epi-
thelial cells [Eger et al., 2005].
NF-kB activation has been associated with
the induction of ZEB1 and ZEB2 expression.
MCF-10A cells stably expressing the NF-kB
subunit p65 (MCF-10A/p65 cells) displayed
elevated levels of expression of ZEB1 and
ZEB2 compared to the parental MCF-10A line
[Chua et al., 2007]. Moreover, in transient
transfection assays, p65 increased ZEB1 pro-
moter activity [Chua et al., 2007]. Induction of
ZEB1 and ZEB2 by NF-kB was also observed
following treatment of MCF-10A cells with IL-
1a or TNFa [Chua et al., 2007]. Thus, ZEB1 and
ZEB2 may serve as key mediators of p65 NF-kB
signaling during EMT.
The bHLH transcription factor Twist is
essential for initiation of mesoderm develop-
ment during gastrulation [Castanon and Bay-
found in breast carcinomas [Watanabe et al.,
736Min et al.
2004; Yang et al., 2004], as well as in several
other cancer types, including melanomas[Hoek
et al., 2004], gastric [Rosivatz et al., 2002], and
prostate cancers [Kwok et al., 2005]. A role for
Twist in cancer metastasis was reported in a
breast cancer model, which suggested that
Twist induces EMT and promotes tumor invas-
ion [Yang etal.,2004].Weinberg and coworkers
ished the metastatic potential of the 4T1 cell
line [Yang et al., 2004]. Twist specifically
mediated intravasation into the systemic circu-
lation rather than survival of tumor cells.
Ectopic Twist expression was sufficient to
induce phenotypic and molecular hallmarks of
EMT in MDCK cells and in immortalized
repressed the E-cadherin promoter and gene
transcription. Of note, an inverse correlation
between high Twist expression and low E-
cadherin levels was seen in human invasive
lobular breast carcinomas [Yang et al., 2004].
Furthermore, elevated Twist expression has
been correlated positively with an aggressive
breast cancer phenotype and poor patient
survival [Hoek et al., 2004; Yang et al., 2004;
Kwok et al., 2005]. A recent study identified
AKT2, a known metastasis gene, as a down-
stream target and functional mediator of Twist
during cell migration and invasion [Cheng
et al., 2007].
Twist is an evolutionary conserved NF-kB
target gene [Wang et al., 1997; Kanegae et al.,
transcriptional target of the NF-kB protein
Dorsal [Jiang et al., 1991; Pan et al., 1991;
Thisse et al., 1991]. Inhibition of NF-kB in mice
byeither expression ofthesuper repressorform
of IkBa or ablation of the IKKa kinase caused a
dramatic impairment of Twist expression, and
tomorphogenetic defects in embryonal develop-
Moreover, Twist can be rapidly induced in
mouse embryonic fibroblasts by TNF-a, and
this induction is essentially absent in cells
lacking the p65 NF-kB subunit [Sosic et al.,
2003]. Interestingly, Pham and coworkers
found that upregulation of Twist by chemo-
therapeutic agents in certain cancers depended
on p65 NF-kB complexes, while in other sys-
tems, c-Rel-containing NF-kB complexes were
sufficient to activate Twist gene transcription
[Pham et al., 2007].
NF-kB IN THE INDUCTION OF
The vimentin (VIM) gene encodes a 56-kDa
cytoskeletal protein that is part of the large
intermediate filament (IF) gene family. Vimen-
tin is the major IF protein found in mesenchy-
mal cells. Because of the abundance of its
expression, it had long been thought to be at
least partially responsible for structural integ-
rity of mesenchymal cells. Thus, it was surpris-
ing that knockout vimentin mice undergo
normal embryonic development into adulthood
and are able to produce offspring with no overt
phenotype [Colucci-Guyon et al., 1994]. How-
ever, further analysis of vimentin ?/? mice
have shown significant impairment of wound
healing in both embryonic and adult animals
[Eckes et al., 2000], likely due to reduced
migratory ability of vimentin ?/? fibroblasts
[Eckes et al., 1998]. These studies have solidi-
fied a functional role for vimentin in cellular
migration in addition to its status as a mesen-
ing the completely dedifferentiated state in
tumor cells that are highly proliferative and
sionofvimentin in breast cancer cells in culture
led to enhanced migration of breast cancer cells
[Hendrix et al., 1996]. Conversely, knockdown
of vimentin with antisense oligonucleotides
reduced cell motility. In vivo expression of
vimentin, however, is a rare occurrence even
in invasive breast cancers. In a recent study
using tissue microarray analysis, vimentin was
found expressed in 21/272 breast cancer cases
and correlated positively with tumor grade
[Korsching et al., 2005]. Most were found to be
Grade 3 invasive ductal carcinomas (19/21) but
the majority (13/21) of these were associated
with the ductal in situ component [Korsching
in EMT in vivo may be more complicated due to
its possible involvement in progenitor cells.
The VIM promoter is comprised of multiple
elements responsible for its transcriptional
regulation. An NF-kB binding site has been
implicated in growth factor responsiveness
[Lilienbaum et al., 1990; Lilienbaum and Pau-
NF-kB and Epithelial Mesenchymal Transition 737
trophic virus-1 (HTLV-1) Tax protein, a well-
known activator of NF-kB [Lilienbaum et al.,
1990]. Tax-induced activation of NF-kB leads to
NF-kB binding to an element on the VIM
promoter located between nucleotides ?239
and ?197 bp as judged by binding, competition
[Lilienbaum and Paulin, 1993]. Overexpression
of the RelB in breast cancer cells induces
vimentin expression, and a more mesenchymal
phenotype [Wang et al., 2007]. Additionally,
overexpression of a constitutively active form
of p65 in MCF-10A breast cancer cells also
results in increased expression of vimentin and
to a more mesenchymal phenotype [Chua et al.,
MMP-2 and MMP-9
Matrix metalloproteases (MMPs) are type IV
collagenases, which increase cellular invasive-
ness and mobility. MMP-2 and MMP-9 are
members of the family of 28 known Zn2þ- and
teases. MMP-2 and MMP-9 degrade compo-
nents of the extracellular matrix including
denatured collagen, native type IV collagen, as
well as collagen V/XI and elastin [Vu and Werb,
2000]. Of note, the rapid degradation of type IV
collagen in the basement membrane allows for
tumor invasion and metastasis. MMP-9, and to
a lesser extent MMP-2, expression has been
correlated with a poor prognoses in a number of
[Davies et al., 1993b; Jones and Walker, 1997;
Miyamoto et al., 2005]. MMP-9 expression in
breast cancer also correlated with advanced
tumor grade in that MMP-9 was found to be
expressed in 11 of 11 grade III tumors [Davies
expression of MMP-9 was found in grade III
breast tumors than in lower grade and benign
tumors or normal mammary tissue [Davies
et al., 1993a]. In a tissue microarray analysis
of 131 patient cancer specimens, high expres-
sion of MMP-9 was associated with shortened
relapse-free survival [Vizoso et al., 2007], con-
sistent with its role as a type IV collagenase
involved in tumor invasion and metastasis
[Jones et al., 1999].
In early animal studies of metastasis, H-Ras-
transformed rat embryo fibroblasts were found
to express MMP-2 and MMP-9 and to display
enhanced metastatic behavior in nude mouse
models [Garbisa et al., 1987]. Addition of tissue
inhibitor of MMP (TIMP) resulted in inhibition
of the MMP-9 (92 kDa type IV collagenase).
transformed rat embryo fibroblasts show a
markedly reduced ability to metastasize to the
lungs of nude mice after repeated recombinant
intraperitoneal injection of TIMP [Alvarez
et al., 1990]. Consistent with these observa-
the highly malignant H-Ras and v-Myc trans-
formed rat embryo 2.10.10 cell line, and ribo-
zyme-mediated degradation of MMP-9 resulted
in reduced metastatic phenotype [Hua and
metastases after subcutaneous or intravenous
type IV collagenase activity [Nakajima et al.,
1987]. Kupferman and coworkers bred female
homozygous mice transgenic for a fragment of
the rabbit MMP-9 promoter (?522 to þ12)
linked to b-galactosidase, with males homozy-
gous for MMTV polyoma middle T antigen, a
transgene that leads to mammary tumorigene-
sis in female mice [Guy et al., 1992]. The
resulting bitransgenic female progeny devel-
oped breast cancer displaying b-galactosidase
expression, as a measure of MMP-9 reporter
activity, only upon the formation of invasive
IV collagenases are induced in metastatic cell
types and are partly responsible for the meta-
NF-kB is responsible for activation of MMP-9
promoter transcription [Himelstein et al.,
1997]. Deletion or mutation of the NF-kB site
at ?599 bp resulted in a threefold reduction in
reporter activity of a human MMP-9-CAT
construct [Himelstein et al., 1997]. Consistent
with these findings, the MMP-9 promoter is
activated in Bcl-2-overexpressing adriamycin-
resistant cells; mutation of the ?599 bp NF-kB
site eliminates Bcl-2-mediated activation of the
promoter. In addition, RelA was found to be the
primary NF-kB factor that binds to this region
[Ricca et al., 2000]. Similar findings were
obtained in SK-N-SH cells, which undergo
spontaneous phenotypic conversion from epi-
thelial to neuroblastic phenotype, which in
these cells represents a more migratory, inva-
sive and tumorigenic phenotype [Farina et al.,
1999]. In transient transfection assays, SK-N-
SH cells displaying an epithelial phenotype
failed to activate an MMP-9-CAT reporter
738Min et al.
construct, while those displaying a neuroblast
phenotype displayed an eightfold elevation in
CAT activity [Farina et al., 1999]. In vitro
footprint analysis revealed two regions on the
MMP-9 promoter relative to the transcriptional
start site that were protected in neuroblastoma
contained a putative NF-kB element and a
putative SP-1 element, respectively. The func-
tional role of the NF-kB site was confirmed
when mutation of the element reduced activity
of the ?670-MMP-9-CAT promoter by 60%
compared to the wild-type control [Farina
et al., 1999]. Thus, NF-kB regulates MMP-9
gene transcription through an element located
approximately 600bases upstreamofthetrans-
criptional start site.
NF-kB can indirectly regulate MMP-2 activ-
ity via control of an enzyme mediating post-
in a precursor form, termed pro-MMP-2. To
produce active MMP-2, the N-terminal pro-
domain must be cleaved, which is done by a
membrane type metalloprotease (MT-MMP)
In dermal fibroblasts, NF-kB can increase
MMP-2 activity by inducing the expression of
MT-MMP rather than by acting directly on the
MMP-2 promoter [Han et al., 2001]. Blocking
osteopontin-induced NF-kB activity with an
IkB-a super-repressor also caused a reduction
in MMP-2 activity in murine melanoma cells
[Philip et al., 2001]. Thus, NF-kB indirectly
regulates MMP-2 activity, which is distinct
et al., 2002].
from the direct transcriptional regulation seen
Consistent with the above data, transgenic
mice overexpressing NF-kB p100/p52 under
control of the b-lactoglobulin promoter display
elevated MMP-9 and MMP-2 activity in the
mammary gland [Connelly
Although the mammary glands did not appear
of mammary cells was found [Connelly et al.,
2007]. Lastly, expression of a constitutively
active IKKe/i in kidney epithelial cells caused
a significant increase in MMP-9 expression
[Boehm et al., 2007], consistent with its role in
invasive phenotype [Eddy et al., 2005]. Con-
versely, osteoclasts deficient in IKKa displayed
muted expression of MMP-9 in response to
RANK activation also consistent with the
requirement for NF-kB in regulation of MMP-9
[Chaisson et al., 2004].
NF-kB is at the center of multiple pathways
Given that NF-kB also plays key roles in innate
immunity, the challenge is to target these
factors effectively in cancer. In a recent review
[Gilmoreand Herscovitch, 2006], 785inhibitors
of NF-kB were described. Introduction of some
of these into the clinic will hopefully enable
more effective treatment of patients with inva-
decrease while expression of vimentin, metalloproteinases
MMP-2 and MMP-9, fibronectin and N-cadherin increases.
NF-kB plays pivotal roles in both facets of this transition, either
The NF-kB family of transcription factors promotes a directly or indirectly. NF-kB induces expression of the master
regulators Snail, Slug, ZIB1, ZIB2 and Twist, which repress
expression of genes encoding epithelial markers such as
E-cadherin. NF-kB also induces expression of mesenchymal
markers Vimentin, MMP-2 and MMP-9, as explained in this
markers are indicated in black font).
NF-kB and Epithelial Mesenchymal Transition739
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