anti-il-20 monoclonal antibody alleviates …...2012/09/19 · il-20 is a proinflammatory cytokine...

IL-20 and oral cancer 1

Anti-IL-20 monoclonal antibody alleviates inflammation in oral cancer and suppresses

tumor growth

Yu-Hsiang Hsu1, Chi-Chen Wei2, Dar-Bin Shieh3, Chien-Hui Chan1 and Ming-Shi Chang1

1Department of Biochemistry and Molecular Biology, 3Institute of Oral Medicine and

Department of Stomatology, College of Medicine, National Cheng Kung University, Tainan,

Taiwan

2Department of Medical Research and Development, Show Chwan Health Care System,

Changhua, Taiwan

Running title: IL-20 and oral cancer

Keywords: IL-20, oral cancer, inflammation

Word count: 5418

Total number of figures: 5

Total number of tables: 0

Conflict of Interest: All the authors declare no competing financial interests.

Footnotes:

Address correspondence and reprint requests to Prof. Ming-Shi Chang, Department of

Biochemistry and Molecular Biology, National Cheng Kung University, College of Medicine,

Tainan 70428, Taiwan. Tel: 886-6-235-3535 ext 5677; Fax: 886-6-274-1694; E-mail:

[email protected]


Abstract

IL-20 is a proinflammatory cytokine involved in rheumatoid arthritis, atherosclerosis, and

osteoporosis. However, little is known about the role of IL-20 in oral cancer. We explored the

function of IL-20 in the tumor progression of oral cancer. IL-20 expression levels in tumorous

and non-tumorous oral tissue specimens from 40 patients with four different stages oral cancer

were analyzed with IHC staining and RTQ-PCR. Expression of IL-20 and its receptor subunits

was higher in clinical oral tumor tissue than in non-tumorous oral tissue. The role of IL-20 was

examined in two oral cancer cell lines (OC-3 and OEC-M1). In vitro, IL-20 promoted TNF-α,

IL-1β, MCP-1, CCR4, and CXCR4, and increased proliferation, migration, ROS production,

and colony formation of oral cancer cells via activated STAT3 and AKT/JNK/ERK signals. To

evaluate the therapeutic potential of anti-IL-20 monoclonal antibody 7E for treating oral cancer,

an ex vivo tumor growth model was used. In vivo, 7E reduced tumor growth and inflammation

in oral cancer cells. In conclusion, IL-20 promoted oral tumor growth, migration, and

tumor-associated inflammation. Therefore, IL-20 may be a novel target for treating oral cancer,

and anti-IL-20 monoclonal antibody 7E may be a feasible therapeutic.


Introduction

With approximately 500,000 new cases annually, squamous cell carcinomas of the head

and neck (HNSCC), are one of the six most common cancers in the world (1). Oral cancer

comprises nearly 30% of all HNSCC. Of these, approximately 90% of cases are squamous cell

carcinomas (2). Oral squamous cell carcinoma (OSCC) is a major health problem worldwide,

and patients have a particularly poor 5-year survival rate (3). The staging of oral cancer is an

important step in defining the natural history and prognosis of the disease, and affects

treatment decisions. Based on the TNM (T: tumor size; N: regional nodal involvement; M:

distant metastasis) classification with various modifications, stages are determined according

to the American Joint Committee on Cancer (AJCC) and the International Union Against

Cancer (UICC) classification system for oral cancer (3). Each stage is a progression through a

primary tumor and progressively nodal, adjacent, and distant metastasis.

Several mechanisms involved in oral cancer have been extensively studied; however, the

poor prognosis may reflect an incomplete knowledge of the mechanisms underlying the

malignant progression of this cancer type. Oral cancer starts with uncontrolled cell

proliferation, and over time becomes able to multiply without limit (immortalization) (4). Next,

oral cancer cells acquire the ability to migrate from their original site, invade nearby tissue, and

metastasize to distant sites. The accumulation of changes in certain molecules results in these

progressive changes from slightly deregulated proliferation to full malignancy.

Prolonged alcohol and tobacco use, betel nut chewing, and infection with the human

papillomavirus (HPV) are known risk factors and have suggested the involvement of chronic

inflammation and oxidative stress in the development of oral cancer (5, 6). Reactive oxygen

species (ROS) are formed in the human oral cavity after they have been exposed to the above

stimulants. ROS induce DNA damage and activate intracellular signals and transcription

factors (7). These alterations in turn affect the production and function of certain molecules for


regulating the cell cycle, growth, differentiation, migration, invasion, and inflammation (8).

Inflammation in the tumor microenvironment has many tumor-promoting effects (9). It aids in

the proliferation and survival of malignant cells, promotes angiogenesis and metastasis,

disrupts adaptive immune responses, and alters responses to hormones and chemotherapeutic

agents (10-12). Among these agents, proinflammatory cytokines, such as IL-lβ, IL-6, and

TNF-α, are essential for regulating the immune response and may have prognostic significance

in cancer. The serum levels of IL-lβ, IL-6, and TNF-α are higher in patients with oral cancer

than in healthy persons, and increased serum levels appear to be correlated with the clinical

stage of the disease (13). Leukocyte trafficking, which is critically regulated by chemokines

and their receptors, shares many of the characteristics of tumor cell infiltration and metastasis.

Increasing evidence suggests a pivotal role for CXCL12/CXCR4 in the establishment of lymph

node and distant metastasis in OSCC (14, 15) by inducing epithelial-mesenchymal transition

(16).

A number of downstream signaling events, such as the Ras/Raf/MAPK, the STAT3, and

the PI3K/AKT/mTOR pathways, contribute to the malignant growth and metastatic potential

of OSCC (17-19). They are often persistently active in oral cancer cells.

IL-20 is a member of the IL-10 family, which includes IL-10, IL-19, IL-20, IL-22, IL-24,

and IL-26 (20), which share 18-25% amino acid identity with IL-10. IL-20 is expressed in

monocytes, epithelial cells, and endothelial cells. It acts on multiple cell types by activating a

heterodimer receptor complex of either IL-20R1/IL-20R2 or IL-22R1/IL-20R2 (21). It is also

involved in various inflammatory diseases, such as psoriasis (22, 23), rheumatoid arthritis (24,

25), atherosclerosis (26), and renal failure (27).

We previously showed (26, 28) that IL-20 not only promoted angiogenesis, but also

increased tumor vascularization. Moreover, IL-20 is regulated by hypoxia and contributes to

brain injury in an ischemic stroke model, which indicates that IL-20 is a promoting factor in the


inflammation-associated disease (29). Most solid tumor cells grow under hypoxic conditions,

which may induce IL-20 overexpression. In addition, HIF-1α is an independent prognostic

marker in oral squamous cell carcinoma and is important in the pathogenesis of oral cancer

(30). Little is known about the involvement of IL-20 in the pathogenesis of oral cancer.

Therefore, we studied IL-20 expression and its biological function in oral cancer cells. We also

evaluated the therapeutic effects of anti-IL-20 monoclonal antibody (mAb) 7E to alleviate or

abrogate key malignant phenotypic characteristics of oral cancer in vitro and in vivo.


Materials and methods

Participants

Forty patients with oral cancer (stage I, n = 10; stage II, n = 10; stage III, n = 10; stage IV,

n = 10) were enrolled in this study. Oral tumor and non-tumorous tissue from these patients was

analyzed. Non-tumorous oral tissue was taken from non-pathological areas distant from tumors

in surgical specimens and histology examinations confirmed that they were non-tumorous.

Biopsies were taken after obtaining informed consents for the study from all participants. The

study was approved by the Ethics Committee of National Cheng Kung University Hospital.

Cell lines

OC-3 cells (31), from a cell line isolated from a Taiwanese patient with OSCC, were

maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum

(FBS) (Life Technologies, Rockville, MD), 2 mM/L of L-glutamine (Life Technologies), 100

μg/mL of streptomycin, and 100 units/mL of penicillin in a humidified 5% CO2 atmosphere at

37°C. OEC-M1 cells (32), from a cell line derived from the gingival epidermal carcinoma of a

Taiwanese patient, were maintained in RPMI 1640 medium.

Immunohistochemistry and immunocytochemistry

Paraffin-embedded-tissue samples were used for immunohistochemistry (IHC) staining

with anti-IL-20 (7E), anti-IL-20R1, anti-IL-20R2, or anti-IL-22R1 mAb (R&D Systems,

Minneapolis, MN) at 4°C overnight as previously described (33). Incubating paraffin tissue

sections with mouse IgG1 isotype (clone 11711; R&D Systems) instead of primary antibody

was the negative control. We used 3 μg/ml as the working concentration for each primary

antibody and for the control mouse IgG1. At least five sections from each patient's specimen

were analyzed and examined by two investigators trained in oral pathology and blinded to

patient details. Immunocytochemical staining of IL-20 and its receptors in OC-3 and OEC-M1

cells was done using the same protocol as described above.


Real-time quantitative polymerase chain reaction (RTQ-PCR)

To examine the oral cancer cell lines and clinical specimens, OC-3 cells, OEC-M1 cells,

oral tumor cells, and non-tumorous oral tissue specimens from 40 patients were analyzed.

Total RNA was isolated (Invitrogen, Carlsbad, CA). Reverse transcription was done with

reverse transcriptase (Clontech, Palo Alto, CA). Human IL-20, IL-20R1, IL-20R2, and

IL-22R1 expression was then amplified on a thermocycler (LC 480; Roche Diagnostics,

Indianapolis, IN), with SYBR Green (Roche Diagnostics) as the interaction agent.

Quantification analysis of mRNA was normalized with hGAPDH used as the housekeeping

gene. Relative multiples of change in mRNA expression was determined by calculating 2−��Ct.

To examine the expression of TNF-α, IL-1β, and MCP-1, OC-3 and OEC-M1 cells were

incubated with IL-20 (200 ng/ml) for the indicated times, and then total mRNA was isolated

and analyzed. The expression levels of these genes were analyzed using RTQ-PCR with

specific primers following the same protocol as described above.

Cell proliferation assay

OC-3 cells (3 × 104) and OEC-M1 cells (4 × 104) were cultured overnight and then

exposed to IL-20 (50-200 ng/ml) for 72 hours in medium containing 1% FBS. To demonstrate

the specific activity of IL-20, 7E (2 μg/ml) was added to the culture system, either alone or

together with IL-20 at a 10:1 concentration ratio (7E:IL-20). OC-3 and OEC-M1 cells

responses to IL-20 stimulation were assessed using 3H-thymidine incorporation assays. Briefly,

after 54 hours of incubation, 1 μCi of 3H-thymidine (NEN Life Sciences, Boston, MA) was

added, and cells were pulse-labeled for 18 hours. The degree of proliferation was determined as

counts per minutes, detected using a scintillation counter (Beckman Coulter, Fullerton, CA).

Cell migration assay

OC-3 and OEC-M1 cells were assayed using a 48-well chamber (modified Boyden

chamber) housing a polycarbonate filter with 8-μm pores (Nucleopore, Cabin John, MD). The


upper wells were loaded with OC-3 (2 × 104) or OEC-M1 (3 × 104) cells. The lower chambers

were filled with IL-20 (100 and 200 ng/ml), 7E (2 μg/ml), or IL-20 plus 7E in medium

containing 0.1% FBS. Medium with 0.1% FBS was used as a negative control. 7E against

IL-20 was used to specifically neutralize the activity of IL-20. The chambers were incubated

for 6 hours at 37°C. Cells adhering to the lower side of the filter were fixed with methanol and

stained in a Giemsa solution (Diff-Quick; Baxter Healthcare, Deerfield, IL) for counting. The

number of OC-3 and OEC-M1 cells on the lower surface of the filter was determined by

counting 12 randomly selected fields under a microscope at 100× magnification. The

experiment was done 3 times using quadruplicate wells, and migration was determined as the

mean of the total cells counted per field.

Flow cytometric analysis

We used fluorochrome, 5,6-carboxy-2,7-dichlorofluorescein diacetate (DCFH-DA) to

measure the intracellular generation of ROS. OC-3 cells (1 × 106) and OEC-M1 (1.5 × 106)

cells were stimulated by the indicated concentrations of IL-20 in serum-free medium for 30

minutes. The OC-3 and OEC-M1 cells were harvested after treatment and stained for 30

minutes at 37°C with 10 μM of DCFH-DA (Molecular Probes, Eugene, OR). After they had

been washed, the cells were kept in the dark and immediately analyzed in a cytometer

(FACSCalibur; BD Biosciences, San Jose, CA). For each analysis, 10,000 events were

recorded.

The expression of CCR4 and CXCR4 on the surface of IL-20-treated OC-3 and OEC-M1

cells was determined using a flow cytometric assay. OC-3 (1 × 106) and OEC-M1 (1.5 × 106)

cells were treated with IL-20 (100 and 200 ng/ml) for 24 hours. To demonstrate the specific

activity of IL-20, 7E (2 μg/ml) was added to the culture system, either alone or together with

IL-20. After they had been treated, the OC-3 and OEC-M1 cells were dissociated into cell

suspension using an enzyme (TrypLE; Invitrogen) and harvested for staining with anti-CCR4


and -CXCR4 monoclonal antibodies (BD Biosciences) at 4°C for 30 minutes. Some OC-3 and

OEC-M1 cells were also left unstained and labeled with nonspecific isotype antibodies as

controls. Subsequently, the stained cells were washed once with PBS containing 1% FBS, and

then subjected to a standard flow cytometric analysis in the cytometer.

Western blotting

OC-3 cells (5 × 105) were stimulated with IL-20 (200 ng/ml) (R&D Systems) for the

indicated time periods. Western blotting used antibodies specific for phosphorylated AKT,

JNK, ERK, and STAT3 (Cell Signaling Technology, Beverly, MA). α-Tubulin was detected

using a specific antibody (Cell Signaling Technology) as an internal control.

Colony formation

OC-3 (2500/well) and OEC-M1 (3500/well) cells were suspended in 0.5% Bacto Agar

and plated onto a layer of 1% Bacto™ Agar in medium containing 5% FBS in 6-well

tissue-culture plates (Corning, Corning, NY). The agar-containing cells were allowed to

solidify overnight at 37°C in a 5% CO2 humidified atmosphere. Additional medium with 5%

FBS with IL-20 (200 ng/ml) was overlaid on the agar, and the cells were allowed to grow

undisturbed. The culture medium was changed every 2 days. To demonstrate the specific

activity of IL-20, 7E (2 μg/ml) was added to the culture system, either alone or together with

IL-20. The cells were cultured for 10 days, fixed in methanol, and stained with Giemsa. Visible

colonies (> 50 cells) were counted with the aid of a dissecting microscope.

Oral cancer-bearing tumor model

All animal experiments were conducted according to the protocols based on the Taiwan

National Institutes of Health standards and guidelines for the care and use of experimental

animals. The research procedures were approved by the Animal Ethics Committee of National

Cheng Kung University. Eight-week-old female SCID mice were used in all experiments. The

left mammary fat pad of each mouse was subcutaneously (s.c.) injected with OC-3 cells (2 ×


106 cells) or OEC-M1 cells (4 × 106 cells). The mice were then randomly assigned to 3 groups

(n = 6 per group), and treated with PBS, 7E (5 mg/kg; s.c.), or mouse IgG (5 mg/kg; s.c.) every

three days for the duration of the treatment regimen. The antibody was injected (s.c.) into the

periphery of the growing tumor in tumor-bearing SCID mice. Healthy controls were not

injected with tumor cells. Fifty days after the tumor cells had been injected, the mice were

killed and their tumor tissue was harvested and weighed. The tumor tissues isolated from the

six mice in each group were fixed in 3.7% formaldehyde for H&E staining. To analyze the

expression levels of IL-20, TNF-α, IL-1β, and MCP-1, the tumor tissue was placed in PBS

solution and homogenized, and then RNA was extracted. Levels of cytokines were detected

using RTQ-PCR with specific primers as described above.

Statistical analysis

Prism 5.0 (GraphPad Software, San Diego, CA) was used for the statistical analysis. A

one-way ANOVA nonparametric test (Kruskal-Wallis test) was used to compare the data

between groups. Post hoc comparisons were done using Dunn's multiple comparison test.

Results are expressed as means ± standard deviation (SD). Significance was set at P < 0.05.


Results

Higher expression of IL-20 and its receptors in oral cancer

To examine whether IL-20 is involved in the pathogenesis of oral cancer, IHC staining

was used to analyze the expression levels of IL-20 in tumorous and non-tumorous oral tissue

specimens from 40 patients with oral cancer. IL-20 and its receptors were strongly stained on

tumor cells of oral tumor tissue, but only slightly stained on endothelial cells in non-tumorous

oral tissue (Fig. 1A). Furthermore, RTQ-PCR showed that transcript levels of IL-20 (Fig. 1B)

and its receptors (Fig. 1C-E) in oral tumor tissue were significantly higher than in

non-tumorous oral tissue samples. Their expression levels also correlated well with the stages

of the oral cancer (Fig. 1B-E). The expression levels of IL-20 and receptor subunits were the

highest in stage IV oral tumor tissue. These data suggested that IL-20 is involved in the

pathogenesis of oral cancer.

IL-20 increased cell proliferation, migration, and ROS production in oral cancer cells

To study the effects of IL-20 on oral cancer cells in vitro, the expression of IL-20 and its

receptors in OC-3 and OEC-M1 cells was analyzed. OC-3 cells were established from an oral

squamous cell carcinoma of a non-smoking long-term areca (betel nut) chewer (31), while

OEC-M1 is a cell line derived from gingival epidermal carcinoma of a Taiwanese patient (32).

RT-PCR and immunocytochemical staining showed that IL-20, IL-20R1, IL-20R2, and

IL-22R1 were all expressed on OC-3 and OEC-M1 cells (Fig. 2A-B) In addition, ELISA was

used to detect the endogenous IL-20 protein secreted in the conditioned media of OC-3 (373.1

pg/ml) and OEC-M1 (214.8 pg/ml) cells. This indicated that IL-20 might target cancer cells

using a paracrine or autocrine mechanism. A 3H-thymidine incorporation assay was used to

analyze the proliferation of oral cancer cells treated with IL-20. IL-20 dose-dependently

increased OC-3 and OEC-M1 cell proliferation after 72 hours of treatment (P < 0.05 compared

with the untreated controls) (Fig. 2C). To confirm the specificity of IL-20-induced proliferation


of OC-3 and OEC-M1 cells, anti-IL-20 mAb 7E was used to neutralize the IL-20-induced

proliferation of OC-3 and OEC-M1 cells. 7E inhibited IL-20-induced cell proliferation (Fig.

2C). To determine whether IL-20 affected cell migration in OC-3 and OEC-M1 cells, a Boyden

chamber assay was used to evaluate the migration ability (Fig. 2D). IL-20 significantly

promoted OC-3 and OEC-M1 cell migration (P < 0.05 compared with the untreated controls),

which was inhibited by 7E (Fig. 2D). Because oxidative stress is vital in the malignant

transformation of oral cancer, we also analyzed the effect of IL-20 on ROS generation in OC-3

and OEC-M1 cells. IL-20 significantly and dose-dependently increased intracellular ROS

production in OC-3 and OEC-M1 cells (P < 0.05 compared with the untreated controls), which

was inhibited by 7E (Fig. 2E). These results suggested that IL-20 increased proliferation,

migration, and ROS production in oral cancer cells, and was critical in the progression of oral

tumor growth.

IL-20 upregulated TNF-α, IL-1β, MCP-1, CCR4, and CXCR4 in oral cancer cells

The molecular pathways of cancer-related inflammation were revealed, which allowed us

to identify new target molecules that might lead to improved diagnosis and treatment.

Therefore, we investigated whether IL-20 is involved in tumor-associated inflammation. We

incubated OC-3 and OEC-M1 cells with IL-20 and used RTQ-PCR to analyze the expression of

proinflammatory cytokines, chemokines, and chemokine receptors. The TNF-α, IL-1β, and

MCP-1 expression significantly increased in IL-20-treated OC-3 (Fig. 3A-C) and OEC-M1

cells (data not shown); however, the expression of other inflammatory mediators, namely, IL-6,

IL-8, MMP-2, MMP-7, and MMP-9, was not affected (data not shown). IL-20 treatment

significantly induced CCR4 and CXCR4 expression on the surface of OC-3 cells (Fig. 3D-E).

Although flow cytometric analysis showed that IL-20 increased CCR4 and CXCR4 expression

on the surface of OEC-M1 cells, they showed no significant difference when the data were

analyzed using ANOVA.


To confirm that IL-20 specifically induced their expression, the OC-3 cells were treated

with IL-20 plus 7E. Flowcytometric analysis showed that 7E had significantly neutralized

CCR4 and CXCR4 expression in co-treated OC-3 cells (Fig. 3D-E). In addition, we found that

IL-20 expression was significantly correlated with CXCR4 expression on the oral tumor tissue

of patients with stage IV cancer (data not shown). To clarify the mechanism that IL-20 uses to

promote tumor progression, the signal molecules of JNK, STAT3, ERK, and AKT were

assessed in IL-20-stimulated OC-3 cells. Western blotting showed that IL-20 promoted the

activation of JNK, STAT3, ERK, and AKT (Fig. 3F). These results indicated that IL-20

induced inflammatory mediators in oral cancer, triggered proliferation-associated signals, and

was a promoting factor for tumor growth.

IL-20 promoted colony formation of OC-3 and OEC-M1 cells

Neoplastically transformed cells show reduced requirements for extracellular

growth-promoting factors, and they are not restricted by cell-cell contact.

Anchorage-independent growth is one of the hallmarks of the malignant transformation of cells.

Therefore, we used a soft agar colony formation assay to monitor the ability of IL-20 in the

anchorage-independent growth of OC-3 and OEC-M1 cells. IL-20 significantly stimulated

OC-3 and OEC-M1 cells to form more colonies than the controls formed, which was inhibited

by 7E (Fig. 4A-B). OC-3 cells co-treated with IL-20 and 7E formed significantly fewer and

smaller (Fig. 4C) colonies in soft agar than did cells treated with IL-20 alone. These results

suggest a significant role for IL-20 in the malignant transformation of oral cancer cells.

7E suppressed in vivo tumor growth and reduced inflammation within the tumor

microenvironment

IL-20 was highly expressed in oral cancer tissue, and it increased oral tumor growth and

inflammation in vitro. 7E has shown specificity and neutralization activity in vitro and in

vivo.(24, 28, 33) Therefore, we analyzed whether 7E reduced tumor growth in vivo. OC-3 cells


were injected into the left mammary fat pads of SCID mice. One day later, the mice were

injected (s.c.) with PBS, 7E (5 mg/kg), or mIgG (5 mg/kg) (n = 6 per group) every 3 days for 50

days. Fifty days after they had been injected with OC-3 cells, all the mice were killed and their

tumors were isolated. Tumors were significantly smaller (Fig. 5A) and weighed significantly

less (Fig. 5B) in the 7E-treated group than in the control mIgG- and PBS-treated groups. H&E

staining showed that immune cell infiltration was reduced in the tumor mass of the 7E

treated-group compared with the mIgG-treated control group (Fig. 5C). Tumor tissue from

each group was then isolated and homogenized to extract RNA. RTQ-PCR assays showed that

IL-20 levels in the tumor were lower in the 7E-treated group than in the mIgG-treated group

(Fig. 5D). In addition, the expression of the inflammation mediators TNF-α, IL-1β, and

MCP-1 was significantly lower in the 7E-treated group than in the mIgG-treated group (Fig.

5E-G). Furthermore, we used OEC-M1 cells to confirm the effect of 7E in tumor-bearing SCID

mice and obtained similar results (Supplemental Fig. 1). These results indicated that 7E

suppressed tumor growth in vivo by reducing IL-20 expression, decreasing other

proinflammatory cytokines, and inhibiting immune cell infiltration.


Discussion

We found that both IL-20 and its receptors were highly expressed in primary,

nodal-involved, and distantly metastatic oral tumor tissue. IL-20 expression was correlated

with the tumor stages in oral cancer patients. All IL-20 receptor subunits were expressed in our

clinical specimens, which suggested that both autocrine and paracrine effects of IL-20 on oral

tumors and contributed to the pathogenesis of oral cancer.

IL-20 directly affected oral cancer cell proliferation by activating proliferation-associated

signals such as JNK, ERK, and STAT3, and migration by activating migration-associated

signals such JNK and ERK. IL-20 also provided a microenvironment for tumor growth and

metastasis by inducing ROS and proinflammatory cytokines, which are essential for the

malignant process from normal epithelium to invasive oral cancer (34). ROS may increase the

inflammatory responses mediated by activating signaling molecules such as ERK and JNK,

and transcription factors like NF-κB. NF-κB regulates many proteins involved in tumor

initiation, tumor expansion, and metastasis promotion in many types of cancers, including oral

cancer (35, 36).

A variety of cytokines, such as IL-1, IL-6, and TNF-α, as well as chemokines, such as

IL-8, CXCL5, CXCL6, CXCL7, CCL5, and CCL20, were upregulated in progressive oral

cancer cells (6, 37, 38) and in patients with oral cancer (13). The effects of CCL and CXCL

chemokines are mediated via their CCR and CXCR receptors. IL-20-treated OC-3 and

OEC-M1 cells increased TNF-α and IL-1β production, which are potent proinflammatory

cytokines for the growth, metastasis, and immune evasion of oral tumors. We also found that

IL-20 induced MCP-1 and CCR4 expression in OC-3 cells. MCP-1(39, 40) and CCR4 (41, 42)

have been associated with several cancers. Therefore, IL-20 may promote leukocyte trafficking,

which augments inflammation, tumor cell infiltration, and metastasis in oral tumors, by such a

novel, undefined mechanism. Several studies (43, 44) have associated CXCR4 and oral cancer.


IL-20 induced more CXCR4 expression on the surface of IL-20-treated OC-3 cells than of the

untreated control group. The involvement of CXCR4 signaling in OSCC was mediated by the

activation of both the ERK and the AKT/PKB pathways (45). Therefore, IL-20-induced

activation of ERK and AKT may cross-link to amplify the CXCR4 signaling for the

chemotaxis of oral cancer cells. 7E treatment to inhibit IL-20-induced CXCR4 expression may

be a potent anti-metastatic therapy against lymph-node and distant metastases in cases of

CXCR4-related OSCC. IL-20 increased the anchorage-independent growth of OC-3 and

OEC-M1 cells, which confirmed that IL-20 was potently involved in the malignant

transformation of oral cells.

IL-20 was upregulated in the tumor tissue of oral cancer patients. In our mouse tumor

model using OC-3 and OEC-M1 cells, we confirmed that IL-20 was highly expressed in the

tumor mass, which is consistent with our clinical findings. IL-20 is a hypoxia-inducible gene

and the hypoxia response element (HRE) has been identified in IL-20 promoter (29). Areca

(betel) nut extract modulates a signaling cascade that induces HIF-1α expression in oral cancer

cells (46). HIF-1α is an independent prognostic marker in OSCC (30). Therefore, one possible

mechanism upregulating IL-20 in the tumor may be the response of oral cancer cells to hypoxia.

HIF-1α binds to the hypoxia response element on IL-20 promoter and activates IL-20

expression. Human HPV infection is a known risk factor in the development of oral cancer (1,

5). Whether HPV regulates IL-20 expression in the pathogenesis of oral cancer is not well

understood. Additional investigations of the regulation between IL-20 and HPV infection are

required.

7E suppressed OC-3 and OEC-M1 tumor growth and tumor weight in vivo. The inhibition

of tumor growth and tumor size in 7E-treated mice correlated with lower IL-20 expression,

which indicated that IL-20 was involved in the progression of oral cancer by regulating tumor

cell proliferation in vivo.


We hypothesize a working model of IL-20 in oral cancer progression. Oral cancer cells or

infiltrated immune cells secrete a large amount of IL-20, which promotes tumor cell

proliferation by phosphorylating ERK, JNK, AKT, and STAT3 in an autocrine/paracrine

manner. The overexpressed IL-20 stimulates ROS production in tumor cells. IL-20-induced

production of proinflammatory cytokines and ROS by tumor cells amplify local inflammation

within the tumor microenvironment. Eventually, all of these tumor-promoting effects

contribute to the proliferation and survival of malignant cells, angiogenesis, metastasis, and the

impairment of immune responses. In addition, the chemoattractant activity of IL-20 further

increases immune cell infiltration, which results in IL-20 overexpression, which, in turn, forms

a positive loop to oral cancer progression. 7E suppresses IL-20-induced tumor growth and

local inflammation in vitro and in vivo, which supports our hypothesis.

We previously showed that IL-20 was induced in response to hypoxia; promoted

angiogenesis in endothelial cells; induced IL-6, IL-8, and MCP-1 expression in several types of

cells; and was a chemoattractant for neutrophils. In the present study, we showed that IL-20

increased the proliferation and migration of oral cancer cells. IL-20 also induced the expression

of TNF-α, IL-1β, and MCP-1, as well as the production of CCR4 and CXCR4, on oral cancer

cells. These properties of IL-20 provide evidence that IL-20 is involved in many phases of

tumor progression and nurtures a microenvironment for tumor cells.

In summary, our findings indicate that IL-20 is involved in oral cancer progression. IL-20

not only directly increased the proliferation, anchorage-independent growth, and migration of

cancer cells, but also generated a microenvironment that facilitated tumor progression by

inducing the production of ROS and of several proinflammatory cytokines and chemokine

receptors. We conclude that IL-20 is a novel target for pharmacological intervention for

inhibiting the tumor growth and metastasis of oral cancer.


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Figure Legends

Figure 1. Higher expression of IL-20 and its receptors in oral cancer. Surgical biopsy

samples from 40 patients with oral cancer were collected. Oral tumor and non-tumorous tissue

from 40 patients was analyzed. (A) IHC staining showed that IL-20 and its receptors were

strongly stained in four different stages of oral tumor samples but only slightly stained on

endothelial cells in non-tumorous oral tissue samples (stage I, n = 10; stage II, n = 10; stage III,

n = 10; stage IV, n = 10). (B-E) The mRNA of the oral tissues was isolated for real-time

quantitative polymerase chain reaction (RTQ-PCR) analysis using IL-20-, IL-20R1-, IL-20R2-,

and IL-22R1-specific primers. Quantification analysis of mRNA was normalized with

hGAPDH as the housekeeping control gene. Differences between the oral tumor and

non-tumorous oral tissue are statistically significant. * = P < 0.05, ** = P < 0.01 versus

non-tumorous oral tissue controls. All experiments were done 3 times with similar results. Data

are from a representative experiment.

Figure 2. IL-20 increased proliferation, migration, and ROS production in oral cancer

cells. (A) The expression of IL-20 and its receptors in OC-3 and OEC-M1 cells was analyzed

using RT-PCR with specific primers. (B) IHC staining showed that IL-20 and its receptors,

IL-20R1, IL-20R2, and IL-22R1, were expressed in OC-3 and OEC-M1 cells. (C) OC-3 (3 ×

104) and OEC-M1 (4 × 104) cells were cultured for 72 hours in 1% FBS with or without IL-20

protein (50-200 ng/ml). Cell proliferation was determined using a 3H-thymidine incorporation

assay. 7E against IL-20 (2 μg/ml) was used to specifically neutralize the proliferative activity

of IL-20. * = P < 0.05 versus untreated controls. # = P < 0.05 versus treatment with IL-20 alone.

Values are means ± standard deviation of triplicate experiments. (D) OC-3 (2 × 104) and

OEC-M1 (3 × 104) cells were incubated for 6 hours in medium containing 0.1% FBS with

IL-20 (100-200 ng/ml), 7E (2 μg/ml), or IL-20 plus 7E. Medium alone was used as a negative


control. 7E against IL-20 (2 μg/ml) was used to inhibit the activity of IL-20. Cell migration was

evaluated using a modified Boyden chamber assay. * = P < 0.05 versus untreated controls

(medium alone). # = P < 0.05 versus treatment with IL-20 alone. Values are means ± standard

deviation of triplicate experiments. (E) OC-3 and OEC-M1 cells were treated with IL-20 for

the indicated concentrations in serum-free medium for 30 minutes, and ROS generation was

measured using flow cytometry. * = P < 0.05 versus untreated controls. # = P < 0.05 versus

treatment with IL-20 alone. Values are means ± standard deviation of triplicate experiments.

Figure 3. IL-20 upregulated TNF-α, IL-1β, MCP-1, CCR4, CXCR4, and activated

intracellular signaling in oral cancer cells. (A-C) OC-3 cells were treated with IL-20 (200

ng/ml) for the indicated times, and the expression levels of TNF-α, IL-1β, and MCP-1 were

analyzed using RTQ-PCR with specific primers. * = P < 0.05 versus untreated controls. All

experiments were done 3 times with similar results. Data are from a representative experiment.

(D-E) OC-3 cells were treated with IL-20 in the indicated concentrations in DMEM for 24

hours. Cells were dissociated into a single cell suspension and harvested for flow cytometry

using anti-CCR4 and anti-CXCR4 monoclonal antibody. * = P < 0.05 versus untreated controls.

# = P < 0.05 versus treatment with IL-20 alone. Values are means ± standard deviation of

triplicate experiments. (F) OC-3 cells (5 × 105) were incubated with IL-20 (200 ng/ml) for the

indicated time periods, and then cell lysates were collected and analyzed using immunoblotting

with specific antibodies against phospho-JNK, phospho-STAT3, phospho-ERK, and

phospho-AKT. α-Tubulin antibody was used as an internal control. Results are representative

of 3 independent experiments.

Figure 4. IL-20 promoted colony formation in vitro. (A-B) OC-3 (2500/well) and OEC-M1

(3500/well) cells were incubated with IL-20 for 10 days, and then a colony-formation assay

was done. The culture medium was changed every 2 days. IL-20 significantly increased the

colony-formation ability of oral cancer cells. 7E (2 μg/ml) was used to inhibit the activity of


IL-20. * = P < 0.05 versus untreated controls. # = P < 0.05 versus treatment with IL-20 alone.

Values are means ± standard deviation of triplicate experiments. (C) IL-20 also increased the

size of colonies in a soft agar assay. Representative photos are shown for each group.

Figure 5. Anti-IL-20 mAb treatment suppressed tumor growth in vivo. OC-3 cells (2 × 106)

were injected (s.c.) into the mammary fat pads of SCID mice. One day later, the mice were

injected (s.c.) with PBS, 7E (5 mg/kg), and mIgG (5 mg/kg) (n = 6 per group) every 3 days for

50 days. (A) Tumor size was measured using a vernier caliper. * = P < 0.05 versus mIgG

controls. ** = P < 0.01 versus mIgG controls. (B) Mice were killed 50 days after they had been

injected with OC-3 cells, and their tumors were collected and weighed. * = P < 0.05 versus

mIgG controls. (C) Tumor sections from each group were stained with hematoxylin and eosin

(magnification: 200×). Representative photos are shown for each group. (D-G) Tumor tissue

samples from each group (n = 6) were isolated at the end of the experiment. The expression of

IL-20, TNF-α, IL-1β, and MCP-1 in the tumor tissue was analyzed using RTQ-PCR with

specific primers. * = P < 0.05 versus mIgG controls. Results are from a representative

experiment. All experiments were done 3 times. Values are means ± standard error of the

mean.

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