Inflammation, Cancer and Chemoresistance: Taking Advantage of the Toll-Like Receptor Signaling Pathway

Inflammation, Cancer and Chemoresistance: Taking Advantageof the Toll-Like Receptor Signaling PathwayRui Chen1,2, Ayesha B. Alvero2, Dan-Arin Silasi2, Gil Mor2

1Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA;2Department of Obstetrics, Gynecology & Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA

Introduction

Substantial evidence indicates that bacterial- and

viral-induced inflammatory process can mediate tu-

morigenesis.1 It has been observed in animal studies

that surgical removal of a primary tumor is often fol-

lowed by rapid growth of previously dormant meta-

stases and lipopolysaccharide (LPS) has been

suggested to be responsible for this effect.2 Indeed,

BALB/c mice receiving a tail vein injection of 4T1

mouse mammary carcinoma cells showed an

increase in lung metastases following LPS injection.3

LPS is recognized by Toll-like receptor (TLR)-4, a

proinflammatory cell surface receptor which is

expressed on cells of the innate immune system as

well as epithelial cells. Following its ligation, TLR4

induces NF-jB activation, cytokine/chemokine pro-

duction and inflammation.4

In humans, chronic infection and inflammation

are considered two of the most important epigenetic

and environmental factors contributing to tumori-

genesis and tumor progression.5,6 Individuals with

ulcerative colitis, a chronic inflammatory disease of

the colon, have a 10-fold higher likelihood of devel-

oping colorectal carcinoma. Similarly, inflammatory

conditions of the liver, such as chronic hepatitis and

cirrhosis are well-established risk factors for the

development of hepatocellular carcinoma.1 Ovarian

endometriosis, a condition which promotes a proin-

flammatory environment within the ovary on a

cyclical basis, predisposes women to specific types

of epithelial ovarian cancers (EOC).7–9 In contrast,

Keywords

Chemokines, chemoresistance, epithelial

ovarian cancer, MyD88, paclitaxel, Toll-like

receptor

Correspondence

Gil Mor, Department of Obstetrics,

Gynecology & Reproductive Sciences,

Reproductive Immunology Unit, Yale

University School of Medicine, 333 Cedar St

FMB 301, New Haven, CT 06520, USA.

E-mail: [email protected]

Submitted September 13, 2006;

accepted October 6, 2006.

Citation

Chen R, Alvero AB, Silasi D-A, Mor G.

Inflammation, cancer and chemoresistance:

taking advantage of the toll-like receptor

signaling pathway. Am J Reprod Immunol

2007; 57:93–107

doi:10.1111/j.1600-0897.2006.00441.x

The association between chronic inflammation and cancer has long been

observed. Furthermore, NF-jB activation and the subsequent production

of cytokines, chemokines, growth factors, and antiapoptotic proteins has

been found to be involved in cancer progression and chemoresistance.

However, the signals inducing NF-jB in cancer cells are still not well

understood. Here, we reviewed the association between chronic inflam-

mation and cancer, the role of NF-jB and its inhibitors as potential anti-

cancer drugs, and Toll-like receptors as possible signal initiators for

NF-jB activation and inflammation-induced carcinogenesis and chemo-

resistance. Furthermore, we propose that, the stimulation of Toll-like

receptors by microbial components and/or endogenous ligands may rep-

resent the initial signal promoting a proinflammatory environment that

will enhance tumor growth and chemoresistance.

REVIEW ARTICLE

American Journal of Reproductive Immunology 57 (2007) 93–107 ª 2007 The Authors

Journal compilation ª 2007 Blackwell Munksgaard 93

chronic use of non-steroidal and anti-inflammatory

agents has been shown to protect against several

tumors.10

Much progress has been made in understanding

how inflammatory cells, through the production of

cytokines and chemokines, drive this process.6

Moreover, there is convincing evidence suggesting

that a proinflammatory profile of cytokines and

chemokines persisting at a particular site can lead to

the development of a chronic disease.11 However,

the molecular mechanism(s) regulating the inflam-

matory response at the tumor microenvironment is

not clearly understood. The objective of this review

is to provide new insights into the mechanisms that

link inflammation and cancer.

Chronic inflammation is associated with tumori-

genesis and cancer progression

Chronic inflammation has long been identified to be

associated with cancer. As early as 1858, Rudolf Vir-

chow had already noticed that the generation of

cancer often happened at the sites of chronic inflam-

mation.12,13 He also hypothesized that chronic

inflammation could promote the proliferation of cells

and thus, the development of cancer. Over the past

15 years, numerous cancers have been shown to be

associated with local chronic inflammation. Chronic

inflammatory bowel diseases, such as chronic ulcera-

tive colitis and Crohn’s disease have strong associ-

ation with colon cancer.6 Similarly, gastric cancer

development has a strong link to chronic Helicobacter

pylori infection and the resulting inflammation.14

Ovarian endometriosis is an established risk factor

for certain types of EOCs.7–9 Other examples include

chronic bronchitis and lung cancer, schistosomiasis

and bladder cancer, papillomavirus infection and

cervical cancer, chronic pancreatitis and pancreatic

cancer, chronic cholecystitis and gall bladder cancer,

and hepatitis virus B and C infection and liver can-

cer.1,15,16 In addition, epidemiologic studies showed

that regular intake of non-steroidal anti-inflamma-

tory drugs (NSAIDs) lowered the risk of developing

several types of cancers.6,17,18

Immune infiltration is a remarkable phenomenon

in tumor development. The infiltrating host leuko-

cytes, such as neutrophils, tumor-associated macro-

phages (TAMs), dendritic cells, eosinophils, mast

cells, and lymphocytes, are present in both the sup-

porting stroma and the tumor sites, forming the

inflammatory microenvironment.1,15 Previously, the

immune infiltrates were thought to help the host

against the developing tumor; however, recent stud-

ies showed that instead of combating the tumor, the

infiltrating cells contribute to cancer growth and

metastasis, as well as immunosuppression.1,15,19 By

analyzing 1919 cases, Menard et al. in 1997, found

that immune infiltration had no association with

survival prognosis of breast carcinoma in patients

older than 40 years of age.20 Furthermore, in previ-

ous studies we found that the infiltrating macro-

phages in breast carcinoma were a main source of

estrogen, which is a major stimulus of cell prolifer-

ation and cancer progression.21 Similarly, Pollard

et al. reported that the infiltration of macrophages

stimulated breast cancer progression and metasta-

sis,18 and knocking out colony-stimulating factor-1

(CSF-1, a cytokine that regulates macrophage differ-

entiation and function) in a mouse strain inhibited

breast cancer growth and spreading. A study on oral

epithelial squamous cell carcinoma revealed that the

level of immune cell infiltration was positively corre-

lated with the level of morphologic and pathologic

transformation from normal to malignant.22

Inflammation, the good and the bad

A major role of the immune system, innate and

adaptive, is to maintain tissue homeostasis by stop-

ping and/or preventing cell damage caused by inva-

ding pathogens and promoting tissue repair

following a lesion. The control of the immune

response is pivotal for preventing damage. The

immune response has to be prompt, efficient and

suppressed once the invasion is removed. In order to

achieve these objectives immune cells exert multiple

effector functions that are continually fine-tuned as

tissue microenvironments are altered. A weak or

inefficient immune response will allow the invasion

and overgrowth of pathogenic microorganisms; on

the other hand, an excessive immune response will

cause tissue damage. The destructive cycles that are

initiated within tissues by failure to appropriately

activate and/or disengage either arm of the immune

system can result in excessive tissue remodeling, loss

of tissue architecture, protein, and DNA alterations

because of oxidative stress and under some circum-

stances, increased risk of cancer development.

The inflammatory response is initially mediated by

cells of the innate immune system (macrophages,

dendritic cells, neutrophils, and NK cells) followed by

a later adaptive immune response (T and B cells)

CHEN ET AL.


94 Journal compilation ª 2007 Blackwell Munksgaard

which responds to the signals originated by the innate

immune system. Among the cells of the innate

immune system, macrophages showed a high degree

of plasticity and a remarkable capacity to differentiate

according to the signals originated at the site of infec-

tion. In response to cytokines and microbial products,

monocytes/macrophages exhibit specialized and

polarized functional properties. According to their

receptors and cytokine production macrophages have

been classified as M1 and M2 macrophages. M1 macr-

ophages are known to produce interleukin (IL)-12,

IL-23, interferon (IFN)-c, IL-18, and tumor necrosis

factor (TNF)-a, and mediate response against intracel-

lular parasites and tumors. In contrast, M2 macroph-

ages have high levels of scavenger, mannose- and

galactose-type receptors, produce VEGF, IL-6, IL-10,

PG, iNOS, and IDO, and have immunoregulatory

functions. M2 macrophages promote tissue repair and

remodeling and are present in established tumors and

may promote tumor growth.23

Many types of human carcinomas are characterized

by abundant infiltration of immune cells, and in many

occasions their presence is not revealed by standard H

& E staining. However, staining of ovarian cancer

samples with CD45, a marker for leukocytes, revealed

an intensive immune infiltrate surrounding cancer

cells or migrating through its stroma (Fig. 1a,b). Inter-

estingly, we found that necrotic centers represent as

origins of the immune infiltrates and from these cen-

ters the cells migrate toward the rest of the tumor

(Fig. 1c,d). The presence of necrotic cells has been

proposed to represent one of the stimuli for macroph-

age migration and differentiation.24 Indeed, contrary

to what would be expected, the presence of necrosis

in the tumor is associated with bad prognosis. Many

of the cellular factors released by the dying cells may

function as potent stimuli to the immune cells, pri-

marily macrophages.24

The innate immune cells are necessary in the pro-

cess of tissue remodeling. They contribute to blood

vessel formation, aid in tissue clean up, and promote

cell proliferation, especially during the process of

wound healing.25 In the tumor microenvironment,

some types of immune cells have been shown to per-

form similar functions: they enhance angiogenesis,

promote cytokine production, and induce immune

tolerance. How are the immune cells ‘educated’ to

promote the neoplastic process? We propose three

stages (Fig. 2): (i) recruitment – via the production of

chemokines [monocyte chemo-attractant protein

(MCP)-1, GROa, IL-8] cancer cells recruit immune

cells to the tumor microenvironment; (ii) education –

via the secretion of cytokines that regulate immune

cells differentiation [IL-6, TNF-a, macrophage migra-

tion inhibitory factor (MIF)] tumor cells polarize

immune cells toward tumor-supporting cells; and (iii)

response – the differentiated immune cells produce

cytokines, hormones, and growth factors at the tumor

microenvironment that promote tumor growth and

create immune tolerance.

(a) (b)

(d)(c)Fig. 1 Immune infiltrate on ovarian cancer

samples. (a) CD45-positive cells (brown) are

observed between cancer cells (blue) on a fro-

zen section of primary ovarian cancer tumor.

(b) Immune infiltrate found in the stroma of a

musine tumor. Note the distribution of the

immune infiltrate (brown CD45-positive cells).

(c and d) Necrotic centers are observed as

potential origins of the immune infiltrates and

from these centers the cells migrated toward

the rest of the tumor.

TLR4-MYD88 SIGNALING IN OVARIAN CANCER



Our studies have focused to understand the

molecular mechanism allowing the tumor to recog-

nize the microenvironment and control the immune

system. Recently, we observed that ovarian cancer

cells express TLRs, a family of membranal receptors

that function as sensors of the immune system. We

proposed that through TLRs, cancer cells might con-

trol the tumor microenvironment and influence the

activity of immune cells.

Cancer progression and Toll-like receptors

signaling

Toll-Like Receptors and Their Ligands

The receptor protein Toll in Drosophila was found in

1996 to be responsible for the organism’s defense

against fungal infections in addition to its role in

dorsoventral pattern establishment during embryonic

development.26 At present, numerous vertebrate and

invertebrate homologs of the Toll receptor (hence

the name TLRs) have been identified. Vertebrate

TLRs belong to a large multigene family consisting of

six subfamilies (Table I).27 There are currently 10

TLRs in humans (TLR1–10), and 12 in mice (Tlr1–13

except Tlr10, which exists as a pseudogene), all of

which are expressed in a large variety of tissues in

adult individuals.28 TLRs play a key role in the innate

immune system, particularly in inflammatory

response against various invading exogenous patho-

gens. TLRs are membrane-bound receptors that recog-

nize receptor-specific pathogen-associated molecular

patterns (PAMPs) of highly conserved pathogenic

components of bacteria, viruses, fungi, and parasites

essential for their survival (Table I).29–34 In addition,

some TLRs can also be activated by endogenous

ligands, as shown also in Table I.32,35,36

It is noticeable that several endogenous TLR ligands

are released from injured cells. HMGB1 is a proin-

flammatory cytokine and is released by immunocom-

petent as well as necrotic cells,24 heparan sulfate, and

polysaccharide fragments of hyaluronan are also

released by injured tissues.37 Therefore, it is not

surprising that tissue injury and the resulting micro-

necrosis-initiated chronic inflammation may lead to

carcinogenesis and the promotion of cancer.38

Moreover, TLR4 can also be activated by the chemo-

therapeutic drug paclitaxel,39–41 which makes it a

potential target for chemoresistance studies.

The Toll-Like Receptor Pathway and its

Expression in Cancer Cells

Most TLRs signal through a common pathway (Fig. 3)

as they possess a common intracellular domain

known as the Toll/IL-1 receptor (TIR) domain.42

Following TLR ligation, it recruits the adapter pro-

tein myeloid differentiation primary response gene

88 (MyD88), which associates with the intracel-

lular domain of the receptor through a TIR–TIR

Immune Cells

chemokines (MCP-1, GROα,IL-8)

(3) Response

(1) Recruitment

(2) Education

cytokines, hormonesand growth factors

Cytokines(IL-6, TNF-α, MIF)

Cancer Cells

Cancer Cells

Proliferation of cancer cells,blockage of apoptosis, andchemoresistance

Fig. 2 Tumor–immune cells interaction. Can-

cer cells recruit immune cells, induce their dif-

ferentiation into tumor-supporting cells and

receive positive feedback from the trans-

formed immune cells. MCP-1, monocyte

chemo-attractant protein-1; Gro-a, growth-

regulated protein alpha; IL-8, interleukin-8;

IL-6, interleukin-6; TNF-a, tumor necrosis fac-

tor-alpha; MIF, macrophage migration inhibi-

tory factor.

CHEN ET AL.



interaction.43–46 In turn, MyD88, through its death

domain (DD) recruits and activates the DD-containing

serine/threonine kinase, IL-1 receptor-associated kin-

ase (IRAK)-4.44 IRAK-4 in turn recruits and phos-

phorylates another kinase, IRAK-1, and the

phosphorylation-activated IRAK-1 binds to the TNF

Table I Toll-Like Receptor (TLR) Classification and Ligands

Subfamily Member

Ligands of human and mouse TLRs

Exogenous Endogenous

TLR1 TLR1 Triacylated lipoprotein (TLR1/TLR2 dimer) –

TLR2 Peptidoglycans, triacylated lipoprotein, diacylated lipoprotein Hsp60, Hsp70, Gp96, HMGB1

TLR6 Diacylated lipoprotein (TLR2/TLR6 dimer) –

TLR10 – –

TLR14 – –

TLR15 – –

TLR3 TLR3 Double-stranded RNA mRNA

TLR4 TLR4 Bacterial lipopolysaccharide, viral fusion protein,

and envelope proteins, paclitaxel

Hsp60, Hsp70, Gp96, fibrinogen, surfactant protein-A,

fibronectin extra domain A, heparan sulfate, soluble

hyaluronan, b-defensin 2, HMGB1

TLR5 TLR5 Flagellin –

TLR7 TLR7 Viral guanosine- and uridin-rich single-stranded RNA, synthetic

antiviral imidazoquinoline compounds and guanosine analogs

mRNA

TLR8 Viral guanosine- and uridin-rich single-stranded RNA, synthetic

antiviral imidazoquinoline compounds and guanosine analogs

mRNA

TLR9 DNA with non-methylated CpG islands Autoimmune chromatin–IgG complex

TLR11 TLR11 Uropathogenic bacteria and a profiling-like protein –

TLR12 – –

TLR13 – –

TLR16 – –

TLR21 – –

TLR22 – –

TLR23 – –

Fig. 3 Toll-like receptor (TLR) signals. Mem-

branal TLRs, such as TLR4, can recognize

external signals, while cytoplasmic TLR3 or

TLR9 may recognize intracellular signals. Fol-

lowing ligation, the majority of TLRs induce

activation of NF-jB and cytokine production in

a MyD88-dependent manner. TLR4, like TLR3,

can also signal in a MyD88-independent man-

ner, which induces the expression of type I

interferons (IFN) and IFN-inducible proteins.




receptor-associated factor (TRAF)-6. The IRAK-1/

TRAF6 complex then dissociates from IRAK-4 and

binds to another protein complex consisting of TGF-b-

activated kinase 1 (TAK1), TAK1-binding protein 1

(TAB1), and TAK1-binding protein 2 (TAB2) through

the interaction between TRAF6 and TAB2. Following

the new association, the membrane-bound IRAK-1 is

degraded and the remaining complex migrates to

the cytoplasm and recruits ubiquitin-conjugating

enzymes (E2) Ubc13 and Uev1A. This cytoplasmic

complex activates the ubiquitin ligase activity of

TRAF6, and the activated TRAF6, together with the

E2 ligases Ubc13 and Uev1A, ubiquitinates target

proteins including TRAF6 itself, which leads to the

cross-phosphorylation and activation of TAK1.47 The

activated TAK1 leads to subsequent downstream acti-

vation of the nuclear factor of kappa light polypeptide

gene enhancer in B cells (NF-jB; early phase NF-jB

activation) and mitogen-associated protein (MAP)

kinase signaling pathways and results in proinflam-

matory response, characterized by the production of

cytokines and chemokines.

Some TLRs (TLR3 and TLR4) can also induce

NF-jB activation via a MyD88-independent manner

through another TIR domain-containing adaptor

molecule, the TIR domain-containing adaptor indu-

cing IFN-b (TRIF).29,48,49 The recruitment of different

signaling molecules by TRIF upon TLR stimulation

can lead to the activation of different downstream

mediators and targets (Fig. 3). The recruitment of

TRAF6 to TRIF can activate NF-jB signaling in a

probably MyD88-dependent pathway-like manner

(late phase NF-jB activation). The association of

receptor-interacting protein (RIP) 1 with TRIF also

activates NF-jB through a yet unknown pathway

(late phase NF-jB activation). What is more, binding

and activating of a non-canonical inhibitor of NF-jB

kinase (IjB kinase, IKK) TANK-binding kinase 1

leads to the phosphorylation and activation of the

transcription factor IFN regulatory factor-3 (IRF-3),

which transcriptionally upregulates IFN-b, a cytokine

that binds to its receptor and induce the expression

of IFN-inducible genes through signal transducer of

transcription 1. Another non-canonical IKK, the

IKKe/IKKi, is also involved in IRF-3 phosphorylation

and activation. Recently, two independent papers

published in Science reported that the production and

signaling of TNF-a was also required in the TRIF-

dependent signaling pathway.50,51 The detailed

mechanism of this pathway, however, is currently

not well understood.

In a recent study, Huang et al. reported the

expression of TLR4 in murine tumor cell lines and

activation of TLR4 signaling in these tumor cells by

LPS-induced tumor evasion from immune surveil-

lance.52 Finally, we described the ubiquituous

expression of TLR4 in human EOC cells and showed

that in a subgroup of EOC cells that express MyD88

(referred from now on as Type I EOC cells),41 ligation

of TLR4 by LPS-induced cell proliferation and

enhanced cytokine/chemokine production. However,

this was not seen in the subgroup of EOC cells that

do not express MyD88 (referred from now on as

Type II EOC cells). Moreover, in Type I EOC cells,

the upregulated chemokine expression upon the

ligation of TLR4 by LPS-induced monocyte migration

(A. B. Alvero and G. Mor in preparation). Further-

more, our in vitro studies demonstrated that cancer

cells may modulate the response of recruited mono-

cytes as demonstrated by a differential response to

LPS from monocytes previously exposed to Type I

EOC cells compared with just serum-free media con-

trol. The type of cytokines that these tumor-educated

monocytes produced is characteristic of a pro-tumor

profile, including IL-6, MCP-1, MIF, and GRO-a.

In summary, these findings suggest that the

inflammatory conditions observed at the tumor

microenvironment may not only originate from the

immune cells, but also from the cancer cells as well.

In addition, cancer cells can ‘‘educate’’ the immune

infiltrate to produce the type of cytokines that will

facilitate tumor growth and metastasis as well as

acquiring immune tolerance (Fig. 4).

Therefore, the inhibition of this pro-tumor envi-

ronment and/or the rescue of the immune cells from

a pro-tumor to an antitumor response may represent

a new strategy for tumor immunology and tumor

therapy as well. In order to achieve this objective it

is necessary to understand the intracellular pathways

mediating many of these effects, and one of the most

important of these pathways is the NF-jB signaling

pathway.

The role of NF-jB in inflammation, carcinogenesis,

and chemoresistance

NF-jB is a Key Transcription Activator in

Inflammation

A key player in chronic inflammation is the nuclear

factor of kappa light polypeptide gene enhancer in

B cells (NF-jB) homodimers or heterodimers. The

CHEN ET AL.



NF-jB dimers are normally formed between the

same or different types of NF-jB family members,

which are Rel homology domain containing pro-

teins, namely RelA (also known as p65), RelB, c-Rel,

p50 (and its precursor p105), and p52 (and its pre-

cursor p100). The NF-jB complex is a key transcrip-

tion activator in the signal transduction pathways in

immune response, and is normally sequestered in

the cytoplasm by its inhibitor, the inhibitor of NF-jB

(IjB). In the canonical NF-jB signaling pathway,

during an immune response, the IKK is activated by

phosphorylation through an upstream kinase cascade

in response to more than 150 different stimuli,53 and

the activation of IKK leads to the phosphorylation

and ubiquitination of IjB, which directs the inhib-

itor to proteosome-mediated degradation. Degrada-

tion of IjB then results in the exposure of the

nuclear localization signal (NLS) of NF-jB as well

as response-specific NF-jB post-translational modi-

fications, such as phosphorylation, acetylation,

ubiquitination, or prolyl isomerization,54 and subse-

quentially its nuclear translocation. In the nucleus,

activated NF-jB acts as a transcription activator and

promotes the expression of a broad panel of immune

response-related genes. Activation of NF-jB is essen-

tial in proinflammatory immune response upon

stimulation by internal (e.g. factors released by nec-

rotic, apoptotic, or pre-cancerous cells, and

cytokines, such as TNF-a and IL-1b) or external (e.g.

components of invading pathogens, such as gram-

negative bacterial LPS and viral double-stranded

RNA) signals, and leads to the production of proin-

flammatory cytokines, chemokines, growth factors,

and the expression of antiapoptotic genes.

NF-jB is Associated with Cancer and

Chemoresistance

Numerous evidence has been reported that links

NF-jB activation and cancer development. Constitu-

tive NF-jB activation is found in most cancer cell

lines as well as numerous types of tumor tissues.55

Biswas et al. reported that activated NF-jB was pre-

sent in 86% of estrogen receptor-negative and

ErbB2-positive breast tumors.56 Moreover, the activa-

tion of NF-jB is often seen in human hepatocellular

carcinoma,57 prostate cancer,58 acute and chronic

myelogenous leukemia,59,60 acute lymphoblastic

leukemia,61 and multiple myeloma.62 The activated

NF-jB transcriptionally upregulates the expression of

many proinflammatory cytokines, such as IL-1, IL-2,

IL-6, and TNF-a; chemokines, such as IL-8, MCP-1,

and GRO-a; growth factors including VEGF and GM-

CSF; matrix metalloproteinases, such as MMP2 and

IL-10

IL-10

IL-6

MCP-1IL-8

T Helper cells

Macrophage

Neutrophil

NK cell

T reg

TNFα

MyD88

MyD88

Inflammation

Tolerance

Fig. 4 Three-steps on tumor-induced toler-

ance. Type I epithelial ovarian cancer cells

‘educate’ macrophages and T reg cells in

order to modulate immune responses and

create a favorable tumor microenvironment to

facilitate tumor growth and metastasis as well

as acquiring immune tolerance.




MMP9; adhesion molecules, such as ICAM-1, VCAM-

1, and ELAM-1; and antiapoptotic proteins including

c-FLIP, c-IAP1, c-IAP2, XIAP, BCL-2 family members,

such as BCL-XL and p53.16,55,63–65 NF-jB also acti-

vates the expression of cyclo-oxygenase-2 (COX-2),

an enzyme required in the synthesis of prostaglandin-

2 (PGE-2), an important proinflammatory medi-

ator.16,18,64,65 The upregulated expression of this

broad panel of cytokines, chemokines, and growth

factors promotes the proliferation of cancer cells

directly (by means of intracrine, autocrine, paracrine,

or endocrine) or indirectly (by working on the

attracted immune infiltrates and the immune

infiltrate-cancer cell communication), whereas the

antiapoptotic proteins further ensure their survival by

protecting them from apoptotic signals and immune

attack.16,55,64,65 Furthermore, the induced chemo-

kines, growth factors, adhesion molecules, and metal-

loproteinases may also serve in NF-jB-mediated

angiogenesis, tumor invasion, and metastasis.18,55,65

The activation of NF-jB, and the resulting upregu-

lation of proinflammatory agents and antiapoptotic

proteins, often bestows cancer cells acquired resist-

ance to chemotherapeutic drugs.41,65 It has been

shown that numerous chemotherapeutic agents are

capable of activating NF-jB, by various known or

unknown mechanisms.

Topoisomerase inhibitors, such as SN38 (a topo-

isomerase I inhibitor) and doxorubicin (a topoisom-

erase II inhibitor), which induce apoptosis in cancer

cells by inhibiting DNA replication, are known to

activate NF-jB by directly stimulating the IKK com-

plex.66

Paclitaxel, a common chemotherapeutic drug that

belongs to the taxane family induces tumor cell

apoptosis by super-stabilizing the microtubules.

However, it is also a ligand of TLR4, and can activate

NF-jB through the TLR signaling pathway.39–41 As

we recently showed, paclitaxel, through the TLR4/

MyD88 pathway can induce cytokine production

and tumor growth.41 Moreover, it can also activate

the AKT survival pathway (through an unknown

mechanism), induce the expression of antiapoptotic

proteins, such as XIAP and BCL2, and lead to the

activation and nuclear translocation of NF-jB.41,67

All these effects may explain why we observed in

Type I EOC cells an increase, in vivo and in vitro, in

tumor growth following paclitaxel treatment. Tumor

cells have hijacked the binding ability of paclitaxel

to TLR4 allowing the tumor cells to overcome its

cytotoxic effect.

Other examples of cytotoxic drugs with non-classi-

cal effects are the microtubulin polymerization

inhibitors vinblastine and vincristine which activate

NF-jB by promoting the phosphorylation and degra-

dation of IjB-a through the increased protein kinase

C activity.68–70 Similarly, etoposide, cisplatin, gemcit-

abine, sodium butyrate, and trichostatin A induce

NF-jB activation by various mechanisms.65 There-

fore, we propose that the activation of NF-jB by a

chemotherapeutic agent leads to boosted cancer cell

proliferation (by the stimulated proinflammatory

microenvironment) and acquired cellular resistance

to apoptosis (by increased antiapoptotic protein

expression), which result in chemoresistance and

remarkably low overall survival rate.

Ligands that Activate NF-jB Promote Inflammation

and Cancer Development

Tumor necrosis factor-a, as its name indicates, when

applied locally in a high dose, can lead to rapid tumor

regression in humans and mice.19,71 However, two

independent groups showed that the TNF-a signaling

pathway was required for carcinogenesis and mice

strains deficient in effective TNF-a signaling were

resistant to the induction of skin cancer.72,73

Tumor necrosis factor-a is a well-studied proinflam-

matory cytokine that is able to activate NF-jB. TNF-asignaling has been described as ‘a split personality’74

or ‘a double-edged sword’75 because it can either lead

to cell proliferation or apoptosis. Binding of TNF-a to

TNF-a receptor 1 (TNFR1) leads to the recruitment of

a complex which consists of proteins including TNF

receptor-associated death domain (TRADD), RIP, and

TRAF2. The intact complex leads to the activation of

NF-jB, which results in inflammation, cell prolifer-

ation, and survival;19,74,76 however, when some

components of the complex are modified post-transla-

tionally through unknown mechanism(s), these com-

ponents dissociate from the cell membrane and form a

new complex which recruits Fas-associated death

domain (FADD), and further recruits and activates

procaspases, such as procaspase-8 and procaspase-10

and triggers the process of apoptosis.19,74 The mechan-

ism that ultimately leads to the decision whether to

induce inflammation or apoptosis is currently

unknown, although it has been suggested that the

decision-making may depend on the concentration of

TNF-a and the length of stimulation.

Based on our recent studies with ovarian cancer

cells we would like to propose that the status of NF-jB

CHEN ET AL.



activation and cytokine production might determine

whether TNF-a will function as pro-apoptotic or

antiapoptotic factor. Thus, in the case of Type I EOC

cells (MyD88-positive, paclitaxel-resistant) character-

ized by the production of high levels of proinflamma-

tory cytokines, the intracellular apoptotic pathway is

blocked by the expression of antiapoptotic proteins,

such as FLIP and IAPs77 and therefore, binding of

TNF-a to its receptor instead of inducing caspase acti-

vation, induces NF-jB activation and enhances the

production of proinflammatory cytokines (Fig. 5a).

On the other hand, Type II EOC cells (MyD88-

negative, paclitaxel-sensitive) which do not produce

cytokines, TNF-a is able to activate the apoptotic

pathway by inducing caspase activation (Fig. 5b).

Besides TNF-a, there are a wide variety of other lig-

ands that can induce NF-jB activation. These ligands

include IL-1b, BAFF, CD40L, Lymphotoxin b, and the

pool of various exogenous and endogenous ligands of

the TLR family.29,54,78 The activation of NF-jB after

Type I EOC ellsTNFR

TNF-α

TRADDRIP

TRAF2

IKKComplex

NF-κB Activation

Cytokines, Chemokines,Growth Factors

Anti-apoptotic Proteins(FLIP, IAPS)

TLR

Ligand

FADD

No apoptosis

MyD88

Pro-Caspase 8

Caspase 8

Caspase 3,7

Type II EOC ellsTNFR

TNF-α

TRADDRIP

TRAF2

No NF-κB Activation

No Cytokine Production

No Anti-apoptotic ProteinProduction

TLR

Ligand(b)

(a)

FADD

Apoptosis

Fig. 5 (a) Differential response to tumor nec-

rosis factor (TNF)-a by epithelial ovarian can-

cer (EOC) cells. Binding of TNF-a to its

receptor in Type I EOC cells induces NF-jB

activation and enhances the production of

proinflammatory cytokines and antiapoptotic

proteins and results in tumor growth. (b) Bind-

ing of TNF-a to its receptor in Type II EOC

cells induces caspase-dependent apoptosis

due to the inability of NF-jB activation.




the binding of these ligands to their receptors induces

a large variety of ligand-specific production of cyto-

kines, chemokines, growth factors, and antiapoptotic

mediators, which then, similar to the effect of TNF-a,

promote cell proliferation and resistance to apoptotic

cell death.

MyD88-Dependent TLR4 Signaling in Epithelial

Ovarian Cancer Cells Promotes Tumor Growth and

Paclitaxel Resistance

Although the link between inflammation, NF-jB,

and cancer progression has been well characterized,

the specific signaling pathway upstream of NF-jB is

currently not well elucidated. As mentioned above,

MyD88 is one of the proteins required in TLR4-medi-

ated early phase NF-jB activation.29 Our previous

studies41 showed that Type I EOC cells constitutively

secrete IL-6, IL-8, GROa, and MCP-1. In response to

LPS stimulation of TLR4, these cells proliferated and

showed marked increase in the secretion of these cy-

tokines via the activation of NF-jB. In contrast, Type

II EOC cells do not constitutively secrete cytokines,

and no changes in cell proliferation or cytokine pro-

duction were observed following treatment with LPS.

The characteristic proinflammatory environment

generated by Type I EOC cells was lost upon the

knockdown of MyD88, suggesting that an active

MyD88-dependent TLR4 signaling is responsible for

the LPS-induced, NF-jB-mediated EOC cell prolifer-

ation and cytokine secretion.

As mentioned above, paclitaxel, a first-line chemo-

therapeutic agent used in the treatment of ovarian

cancer, is a known TLR4 ligand.39–41 Thus, we also

determined if paclitaxel treatment would induce the

same response in Type I EOC cells as was seen with

LPS. Our results showed that similar to LPS, paclitaxel

also induced NF-jB activation, increased secretion of

proinflammatory cytokines, and proliferation of Type

I EOC cells. In contrast, no cytokine secretion was

observed in Type II EOC cells, and more importantly,

they underwent apoptosis in response to paclitaxel.

These results suggest that an activated MyD88-

dependent TLR4 signaling pathway to NF-jB confers

EOC cells the ability to promote a proinflammatory

environment and the development of paclitaxel

resistance. It is noticeable of the specificity of the

signaling pathway to paclitaxel, as treatment of Type I

EOC cells with carboplatin, a chemotherapeutic drug,

which is not a TLR4 ligand, did induce neither prolif-

eration nor cytokine production. The mechanism that

regulates MyD88 expression in these two types of

cells, and the correlation of MyD88 expression and

clinical response to paclitaxel in patients with EOC;

however, still remains to be determined.

Moreover, endogenous ligands of TLR4, such as

HMGB1, heparan sulfate, and polysaccharide frag-

ments of hyaluronan, which are released from the

damaged tissue and necrotic cells, may also contribute

to cancer progression and paclitaxel resistance.32,35,36

NF-jB Inhibitors as Potential Agents for Cancer

Therapy

The establishment of the role of NF-jB in cancer pro-

gression and chemoresistance opened great interest in

NF-jB inhibitors as potential means of cancer ther-

apy. Because NF-jB activation depends on the phos-

phorylation and activation of IKK and the subsequent

phosphorylation and degradation of IjB, the inhibi-

tion of NF-jB activation can be achieved at three lev-

els: (i) inhibition of IKK activity; (ii) prevention of

IjB degradation; and (iii) direct inhibition of NF-jB.

In colitis-associated cancer mouse model, Greten

et al. showed that knocking-out IKKb in either

intestinal epithelial cells or infiltrating macrophages

reduced the chance of tumorigenesis by 80% and

50%, respectively.79 Likewise, regular intake of

NSAIDs, which inhibits IKK80–83 and COX-2 activ-

ity,84 lowered the risk of developing several types of

cancers, including colon, rectal, gastric, and esopha-

geal cancers.6,15,17,18 Other agents that could inhibit

IKK activity include IKKa and IKKb NEMO-binding

domain-targeting short peptides,85 NEMO oligomeri-

zation-targeting peptides,86 and natural and bioavail-

able inhibitors of IKKb, such as flavonoids,

curcumin, cyclopentenone prostaglandins, BMS-

345541, PS1145, SC-514, and SPC839.16,82,87

IjB is another possible target for NF-jB inhibition.

Upon stimulation, the activated IKK phosphorylates

IjB, which leads to its ubiquitination and 26S proteo-

some-mediated degradation and the release of NF-jB.

Therefore, proteosome inhibitors become of particular

interest as potential NF-jB inhibitors. Hideshima et al.

reported that the proteosome inhibitor bortezomib

(PS-341) inhibits the proliferation of multiple myel-

oma cells completely by preventing IjB degradation.88

Bortezomib also sensitized chemoresistant colorectal

cell lines,89 non-small cell lung cancer cell lines,90 and

pancreatic cancer xenografts91,92 to chemotherapeutic

drugs, such as irinotecan, sodium butyrate, and gem-

citabine. Other proteosome inhibitors, such as

CHEN ET AL.



cyclosporine A, lactacystin, HIV-1 protease inhibitors

ritonavir and saquinavir, and peptide aldehydes

MG101, MG132, and MG115 can also abrogate NF-jB

activation by suppressing proteosome-mediated IjB

degradation, thus qualify themselves as potential anti-

cancer agents.65,82 Another group of inhibitors of IjB

inhibit its phosphorylation thereby prevent its degra-

dation. This group of compounds include BAY11-

7082 and BAY11-7085, which are able to enhance the

level of anticancer drug-induced apoptosis in colon,

esophageal, ovarian, and non-small cell lung cancer

cell lines, as well as multiple myeloma cell lines.93–97

A third group of NF-jB inhibitors that work through

IjB upregulate the expression of IjBa at the transcrip-

tion level and therefore inhibit NF-jB activation, as

represented by glucocorticoids.98 Another way of

inhibiting NF-jB activation through IjB is to intro-

duce dominant-negative non-degradable IjB into the

cells by transgenic methods, which had been proved

to sensitize the human fibrosarcoma cells to apoptosis

induced by ionizing radiation or daunorubicin,99 and

reverse the TNF-a-mediated proliferation of colon and

mammary cancer cells to TRAIL-dependent apop-

tosis.100 Last but not least, blocking the activity of the

ubiquitin ligases that are specific for IjB ubiquitina-

tion may also be a possibility.

In contrast to the previous two classes of NF-jB

inhibitors, compounds that can directly interact with

NF-jB molecules and disrupt their function are not

that well developed. This class of inhibitors include

compounds that block the nuclear translocation of

NF-jB, such as FK506,101 benzyl isocyanate,102 and

lycopene;103 compounds that interfere with the

interaction of NF-jB with nuclear proteins-like

andrographolide;104 and compounds that suppress

NF-jB-DNA binding, e.g. guggulsterone105 and

sesquiterpene lactones.106

Conclusion

Chronic inflammation has long been associated with

carcinogenesis and tumor progression. The activation

of NF-jB, which is seen in most cancer cells, play a

key role in tumor initiation, progression, metastasis,

and chemoresistance by mediating the production of

a large variety of proinflammatory cytokines, che-

mokines, growth factors, collagenases, and antiapop-

totic proteins.65 TLRs are among the major activators

of NF-jB and are the front-line receptors in response

to microbial infection to mount an inflammatory

response. However, the activation of NF-jB through

the stimulated TLRs in local chronic inflammation

may serve as an initiator and give the infected or

injured cells a second chance to evolve into can-

cer cells and proliferate out of control (Fig. 6). In

addition, it may also confer the cancer cells ability

to abduct support from the immune system and

acquired resistance to chemotherapeutic agents, such

as paclitaxel. Further study of the role of MyD88-

mediated TLR signaling in cancer progression and

chemoresistance may shed new light on cancer pre-

vention and provide new targets for cancer therapy.

Normal Cell

Immune Cells

Infection/Injury

Cancer Cell

Cancer Cell

PrecancerousCell

TLR TNFR

TLRTNFR

NF-κB Activation

NF-κB Activation

Proliferation, Anti-Apoptosis

Proliferation, Anti-Apoptosis

ChemokineProduction

Cytokin

e

Product

ion

Cytokines

Growth Factors

Recruitment

Education

SupportiveResponse

Cancer Cell ProliferationAcquisition of Chemoresistnce

Ligand(exogenous orendogenous)

Ligand(exogenous orendogenous)

TNF-α

TLRTNFR

TNF-α

Cancer Cell

Fig. 6 Link between Toll-like receptor (TLR),

inflammation, and carcinogenesis. Toll-like

receptor, through NF-jB activation induces

the production of inflammatory cytokines

which could enhance neoplastic transforma-

tion, lead to immune tolerance and chemore-

sistance.




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