author manuscripts have been peer reviewed and …...- 1 - enhancement of human cancer cell motility...

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Enhancement of human cancer cell motility and invasiveness by anaphylatoxin C5a via

aberrantly expressed C5a-receptor (CD88).

Hidetoshi Nitta1,2, Yoshihiro Wada3*, Yoshiaki Kawano3*, Yoji Murakami3, Atsushi Irie4,

Keisuke Taniguchi7, Ken Kikuchi6, Gen Yamada8, Kentaro Suzuki8, Jiro Honda3,

Masayo Wilson-Morifuji5, Norie Araki5, Masatoshi Eto3, Hideo Baba2 and Takahisa

Imamura1

* these authors contributed equally to this work

1Department of Molecular Pathology, 2Department of Gastroenterological Surgery,

3Department of Urology, 4Department of Immunogenetics,

5Department of Tumor Genetics and Biology, Faculty of Life Sciences, Kumamoto

University, 6Medical Quality Management Center, Kumamoto University Hospital, and

7Department of Pharmacology, Mitsubishi Chemical Medience Corporation, Kumamoto,

and 8Department of Genetics, Institute of Advanced Medicine, Wakayama Medical

University, Wakayama, Japan

Research. on April 12, 2020. © 2013 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 3, 2013; DOI: 10.1158/1078-0432.CCR-12-1204

http://clincancerres.aacrjournals.org/

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Running title: C5a-C5aR axis enhances cancer cell motility and invasion

Key words: C5a, C5a receptor, migration, invasion, MMP

This work was supported in part by a Grant-in-Aid for Scientific Research (B) from the

Japanese Ministry of Education and Science (Grant No. 18390125 to T.I.).

Correspondence and reprint requests should be addressed to T. Imamura

Department of Molecular Pathology, Faculty of Life Sciences, Kumamoto University,

1-1-1 Honjo, Kumamoto 860-8556, Japan

Tel: +81-96-373-5306; Fax: +81-96-373-5308; E-mail: [email protected]

There is no conflict of interest.

Word count: 4447; five figures and one table, four supplementary figures




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Translational Relevance

The anaphylatoxin C5a is a product of complement system activation that occurs in the

cancer microenvironment, however, the role of C5a generated in cancer tissues is

largely unknown. We found C5a-receptor (C5aR) expression in cancer cells of human

cancer tissues samples. Moreover, by Matrigel chamber assay and nude mouse skin

implantation we showed C5a-elicited enhancement of C5aR-expressing cell invasion

through motility activation and matrix metalloproteases (MMP) release. Indeed,

anti-C5aR antibody and MMP inhibitor GM6001 suppressed C5aR-expressing cancer

cell invasion enhanced by C5a. These results illustrate a novel activity of the C5a-C5aR

axis in cancer and suggest that it might be beneficial to target this signaling pathway for

cancer therapy. Furthermore, since C5aR expression was seen in adenocarcinoma,

squamous cell carcinoma and transitional cell carcinoma, C5a-C5aR-targeting therapy

may work for those different types of cancer.




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Abstract

Purpose: The anaphylatoxin C5a is a chemoattractant that induces leukocyte

migration via C5a receptor (C5aR). There is emerging evidence that C5a is generated in

the cancer microenvironment. We therefore sought C5aR expression and a direct

influence of the C5a-C5aR axis on cancer cells.

Experimental Design: C5aR expression was investigated in human cancer tissues

and cell lines. Effects of C5a stimulation on cancer cells were studied by cytoskeletal

rearrangement, time-lapse analysis, Matrigel chamber assay and invasion in nude mouse

in a comparison of C5aR-expressing cancer cells with control cells.

Results: C5aR was aberrantly expressed in various human cancers. Several cancer

cell lines also expressed C5aR. C5a triggered cytoskeletal rearrangement and enhanced

cell motility 3-fold and invasiveness 13-fold of C5aR-expressing cancer cells. Such

enhancement by C5a was not observed in control cells. Cancer cell invasion was still

enhanced in the absence of C5a concentration gradient and even after the removal of

C5a stimulation, suggesting that random cell locomotion plays an important role in

C5a-triggered cancer cell invasion. C5a increased the release of matrix




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metalloproteinases (MMPs) from cancer cells by 2 to 11-fold, and inhibition of MMP

activity abolished the C5a enhancing effect on cancer cell invasion. Compared with

control cells, C5aR-expressing cells spread 1.8-fold more broadly at implanted nude

mouse skin sites only when stimulated with C5a.

Conclusions: These results illustrate a novel activity of the C5a-C5aR axis that

promotes cancer cell invasion through motility activation and MMP release. Targeting

this signaling pathway may provide a useful therapeutic option for cancer treatment.




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Introduction

The complement system is a biochemical cascade involved in immune responses

(1). Previous reports showed that the complement system is activated on cancer cells in

both an animal model (2) and in tissue specimens (3). It was initially suggested that the

complement system might be involved in cancer immune surveillance by its direct

cytolytic effect (3) and the sensitization of cancer cells to the immune effector cells via

release of chemoattractants (4). However, cancer cells seem to evade the complement

attack by expressing either soluble or membrane-associated regulators of complement

e.g. CD55, which protects cancer cells from complement-dependent cytolysis (5, 6) and

anti-cancer immune responses (7, 8). Thus, the complement system in cancer tissues

does not seem to lead to cancer cell eradication.

Anaphylatoxin C5a is an N-terminal 74 amino acid fragment of the α-chain of the

complement fifth component (C5), and is well known to act as a leukocyte

chemoattractant and inflammatory mediator (9, 10). C5a is released by C5-convertase

formed during the process of complement system activation (11), possibly triggered in

response to cancer cells (3). Other C5a producing pathways include C5 cleavage by




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thrombin (12), the ultimate product of the coagulation reaction. This cascade reaction

can be triggered by tissue factor, which is expressed in a wide range of cell types

including cancer cells (13). C5a is also generated by serine proteases from activated

phagocytes (14), which frequently accumulate in cancer tissues. These findings lead us

to the idea that C5a is also likely to be generated in the cancer microenvironment.

C5a activities are mediated by its binding to the membrane-associated C5a receptor

(C5aR; CD88) which was originally identified in leukocyte cell lines (15). C5aR has

since been reported to be expressed in other types of cells such as vascular endothelial

cells, mesangial cells and renal proximal tubular cells. Further studies have revealed

that C5aR expression is also inducible in epithelial cells by inflammatory and infection

stimuli (16). Regarding cancer cells, functional C5aR expression was shown in a human

hepatoma cell line HepG2 (17), whereas normal hepatocytes lack in its expression (16).

These suggest that expression of C5aR is induced in cancer cells as a consequence of

malignant transformation.

C5a and chemokines are chemoattractants, and a body of evidence indicates that a

network of chemokines and their receptors influences the development of primary




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cancers (18-22). Recently, C5a was reported to recruit myeloid-derived suppressor cells

for suppressing the antitumor CD8+ T cell response (2, 23), suggesting its indirect role

in fostering cancer cells by protecting them from the antitumor CD8+ T cells. However,

the direct biological role of C5a-C5aR system in cancer cells is largely unknown. In this

study, we investigated the expression of C5aR in cancer cells of various origins and

analyzed its impact on cancer cell motility and invasiveness upon C5a stimulation.

Materials and Methods

Cell lines.

The human bile duct cancer cell lines MEC and HuCCT1 and the human colon cancer

cell lines HCT15, COLO205, and DLD1 were provided by the Cell Resource Center for

Biomedical Research Institute of Development, Aging, and Cancer, Tohoku University

(Sendai, Japan). Human bile duct cancer cell lines SSp-25 and RBE were obtained from

the Riken Cell Bank (Tsukuba, Japan). Human colon cancer cell lines HCT116 and

SW620 were gifts from Dr. B. Vogelstein, Johns Hopkins University, and Dr. Kyogo Ito,

Kurume University respectively. Cells were cultured in RPMI 1640 or DMEM




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supplemented with 10% fetal bovine serum (FBS), penicillin (40 U/mL), and

streptomycin (40 µg/mL) and were maintained at 37°C in 5% CO2.

Tissue samples, immunohistochemistry and retrospective analysis.

Cancer tissue samples were obtained by surgical resection or core needle biopsy in

Kumamoto University Hospital, and usage of those samples for this study was approved

by the internal ethics committee. Deparaffinized 2-µm-thick sections were pretreated

with 0.3% H2O2 in methanol for 20 min, followed by Protein Block, Serum-Free (Dako

Cytomation, Glostrup, Denmark) treatment for 20 min. Sections were incubated with

the primary antibody against C5aR (2 µg/mL; Hycult Biotechnology, Uden, the

Netherlands) at 4°C overnight, and subsequently stained using EnVision+ solution

(Dako Cytomation) and 3,3’-diaminobenzidine tetrahydrochloride solution containing

0.006% H2O2, according to the manufacturer’s instructions. Nuclei were counterstained

with hematoxylin. Retrospective analysis was performed on 42 intrahepatic

cholangiocarcinoma patients who had undergone liver resection from May 2000 to

November 2009. The relationship between cancer cell C5aR expression and vascular




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invasiveness was investigated and analyzed by Fisher’s exact test.

RT-PCR.

RNA was isolated from cancer cells using the Qiagen RNAeasy kit (Qiagen, Valencia,

CA). cDNA was synthesized from extracted RNA using the RNA PCR kit AMV (Takara,

Shiga, Japan), according to the manufacturer’s instructions. PCR was performed using

TaKaRa Ex Taq HS and primers (sense 5’-CGGGAGGAGTACTTTCCACC-3’ and

anti-sense 5’-CTACACTGCCTGGGTCTTCTG-3’ for human C5aR, and sense

5’-CATCCACGAAACTACCTTCAACT-3’ and anti-sense

5’-TCTCCTTAGAGAGAAGTGGGGTG-3’ for β-actin) under the following

conditions: 36 cycles for C5aR and 32 cycles for β-actin, 30 s at 94°C, 30 s at 58°C, and

30 s at 72°C. PCR products were resolved by electrophoresis using 1% agarose gels and

were visualized by ethidium bromide staining.

Immunoblotting.

To detect C5aR, cell lysates obtained from bile duct or colon cancer cells were analyzed




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by SDS-PAGE under reducing conditions using 10% polyacrylamide gels and were

transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon Transfer

Membranes; Millipore). After blocking with 5% fat-free milk, the membranes were

incubated with anti-human C5aR rabbit IgG (1000-fold dilution; Santa Cruz

Biotechnology, Santa Cruz, CA) or polyclonal anti-actin rabbit IgG (500-fold dilution;

Santa Cruz Biotechnology). This was followed by incubation with HRP-conjugated

anti-rabbit IgG goat antibody (1000-fold dilution; Amersham Biosciences, Blauvelt,

NY) and bands were visualized via enhanced chemiluminescence (ECL), according to

the manufacturer’s instructions.

Establishment of C5aR stably expressing HuCCT1 cells.

Full-length human C5aR cDNA of 1053 bp was amplified by PCR using human

macrophage cDNA library and subsequently subcloned into the

pENTR/D-TOPO-vector (Invitrogen, Carlsbad, CA). After confirming the sequence, the

cDNA was inserted into pCAG-IRES-puro vector using the Gateway system

(Invitrogen). The purified plasmid was transfected into HuCCT1 cells using




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Lipofectamine 2000 (Invitrogen). After 48 h, medium was replaced with selection

medium supplemented with puromycin (1 µg/mL) to be cultured for 2 weeks.

Puromycin resistant cells were collected and were subjected to cell sorting by FACS

Vantage to isolate those cells highly expressing C5aR (designated HuCCT1/C5aR).

HuCCT1 cells transfected with empty-pCAG-IRES-puro vectors were used as the

control (HuCCT1/mock).

Flow cytometric analysis.

MEC, HuCCT1/mock or HuCCT1/C5aR cells were treated for 30 min with a murine

monoclonal FITC-conjugated anti-C5aR antibody (Serotec Ltd., Oxford, UK), or a

FITC-conjugated isotype matched control antibody (Serotec Ltd.), followed by washing

with PBS twice. C5aR antigen was quantified by FACScan (BD Biosciences).

Immunofluorescence analysis.

Filamentous actin (F-actin) formation was visualized as previously described (24). Cells

were seeded at a low density on glass coverslips and were incubated for 24 h. After 2 h




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serum starvation, cells were stimulated with 100 nM human C5a (Sigma, St Louis, MO)

for the stated time periods. Cells were then fixed in 4% paraformaldehyde,

permeabilized in 0.2 % Triton X-100 for 5 min, and were incubated with 5 U/mL Alexa

488-phalloidin (Molecular Probes, Eugene, OR) for 40 min, followed by washing with

PBS. Images were obtained and processed by FluoView 300 Laser Scanning Confocal

Microscope (Olympus, Melville, NY).

Time-lapse video analysis.

Cells (1 x 104/well in RPMI1640) were cultured in a 24 well-glass bottom plate (Iwaki,

Funabashi, Japan) for 24 h. After addition of C5a (final concentration: 100 nM), cells

were maintained at 37˚C in 5% CO2 within the chamber set under the camera during the

observation. Images were obtained using 20X UPlan SApo objective (Olympus IX81,

Tokyo, Japan). The camera, shutters, and filter wheel were controlled by MetaMorph

imaging software (Molecular Devices, Sunnyvale, CA), and images were collected

every 10 min with an exposure time of 50 ms. Cell migration distance was measured by

tracing individual cells using MetaMorph imaging software according to the




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manufacturer’s instructions.

Invasion assay in vitro.

To assess invasion of cancer cell lines in vitro, BioCoat Matrigel invasion chambers

were utilized (24-well plate, 8-µm pore; BD Biosciences) (25). HuCCT1-derived

(3.75×104 cells), or MEC (7.5×104 cells) cells were suspended in serum-free RPMI

1640 then seeded into the upper chamber. RPMI 1640 supplemented with either C5a or

carrier solution (PBS) was placed in the lower chamber. To block C5aR-mediated

signaling, anti-human C5aR rabbit IgG (10 µg/mL) or nonspecific control IgG

(10 µg/mL) was added to the cell suspension before seeding. For analyzing the effect of

discontinuous stimulation with C5a, cells were cultured at 37°C in serum-free

RPMI 1640 supplemented with C5a for 12 h (HuCCT1-derived cells) or 24 h (MEC) at

indicated concentrations. Cells were then washed with serum-free RPMI 1640 and were

seeded into the upper chamber. RPMI 1640 containing 10% FBS was set in the lower

chamber. Chambers were incubated for 24 h (HuCCT1-derived cells) or 36 h (MEC) at

37°C. Cells on the upper surface of the filter were removed with a cotton wool swab,




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and cells that migrated to the lower surface were fixed in 100% methanol and were

stained with 1% toluidine blue. Invaded cells were counted in five power fields (×20).

The invasion-enhancing effect was shown as the ratio of cell invasion by C5a

stimulation versus PBS controls. To determine whether MMPs were involved in

C5a-elicited cancer cell invasion, GM6001 (5 µM; Merck, Darmstadt, Germany) was

added to the cell suspension when cell invasion activity of 100 nM C5a was measured.

For checkerboard analysis for C5a cancer cell invasion activity, various concentrations

of C5a were added to the HuCCT1/C5aR cell suspension in the upper chamber together

with the lower chamber, and cell invasion was assessed as described above.

Invasion assay in vivo.

HuCCT1/mock and HuCCT1/C5aR were incubated in serum-free medium in the

presence or absence of C5a (10-7 M) at 37°C for 12 h. This was followed by labeling

with CellTrackerTM Orange CMTMR (20 µM) or CellTrackerTM Green BODIFY

(25 µM) (Molecular Probes) for HuCCT1/C5aR and HuCCT1/mock, respectively, at

37°C for 45 min. After washing with serum-free medium, HuCCT1/mock cells and




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HuCCT1/C5aR cells were equally mixed to create a concentration of 3×107 cells/mL

each. The cell mixture (50 µL) was injected intradermally into 7-week-old BALB/cA

Jc1-nu/nu mice (CLEA Japan, Tokyo). After 1, 2, or 3 days, the nude mice were

sacrificed by cervical dislocation, and the cell injection sites including surrounding

tissues were excised to prepare frozen sections in liquid nitrogen. Labeled cells in

4-µm-thick sections were observed with a fluorescence microscope (BIOREVO;

KEYENCE, Osaka, Japan). To quantify the distribution of HuCCT1-derived cells,

regions of fluorescent dots of labeled cells were encircled (Fig. 6A) then the area of

each region was measured using an imaging processor (VH-Analyzer; KEYENCE). The

ratio of the distribution area of HuCCT1/C5aR versus HuCCT1/mock was calculated.

Some endogenous green fluorescence background was observed in mice skin, therefore

these spots were avoided and cancer cells were specifically encircled, which was

confirmed by observation of the adjacent section HE-stained. This experiment was

performed according to the criteria of animal experiments of the Kumamoto University

Animal Experiment Committee.




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Measurement of MMP concentration in culture supernatant of cells.

MMP concentration in supernatant of cancer cells stimulated with or without C5a was

measured using the Quantibody Human MMP Array 1 kit (RayBiotech, Norcross, GA).

Culture supernatant was taken from MEC (1×106 cells) or HuCCT1/C5aR (5×105 cells)

grown in a 6-well plate for 24 h in the presence or absence of C5a (100 nM). The

supernatant was diluted at 1:3 with PBS then MMP concentrations were determined

according to the manufacture’s instructions.

Statistics.

Statistical analyses were performed using the unpaired Student’s t-test. Values are

expressed as means ± SD and experiments were performed in triplicate, unless

otherwise stated.

RESULTS

Aberrant expression of C5aR in human cancer cells.

We first investigated C5aR expression in human cancer specimens from 225




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patients by immunohistochemistry. C5aR expression was observed in cancer cells from

all the organs examined and in all the three cancer cell types, squamous cell carcinoma,

adenocarcinoma and transitional cell carcinoma (Fig. 1A and supplementary Fig. 1A).

Generally, C5aR was robustly expressed in a significant proportion of cancer samples.

On the other hand, null or only faint reaction of C5aR immunohistochemistry was

observed in their normal counterparts (Fig. 1, A and supplementary Fig. 1B) except

kidney tubular epithelial cells (Supplementary Fig. 1B), which is in line with the

previous report (16). Percentage of C5aR-positivity in cancer cases varied among

organs. In colon, bile duct, kidney and prostate carcinomas, more than 50% of cases

examined were C5aR-positive (Fig. 1B). In bile duct-derived cancer in the liver, C5aR

was positive in 26 patients. Among them, vascular invasion was found in 18 patients,

whereas vascular invasion was seen in only 4 cases out of 16 C5aR-negative patients.

This result indicates a significant relationship between cancer cell C5aR expression and

vascular invasiveness (p = 0.010 by Fisher’s exact test). Since vascular invasion of bile

duct cancer is closely linked to metastasis and prognosis (26), C5aR expression may

correlate with those clinical endpoints. Next, we examined a panel of cancer cell lines




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for C5aR expression. RT-PCR revealed that several human cancer cell lines originated

from bile ducts (MEC and RBE) and colon (HCT15, COLO205, and HCT116)

expressed C5aR mRNA (Fig. 2A). Out of these cell lines, those except RBE also

expressed C5aR protein (Fig. 2B). The localization of C5aR on the cell-surface was

shown by flow cytometry (Fig. 2C). These results suggest that aberrant C5aR

expression observed in human cancer specimens is actually conserved in some human

cancer cell lines. It is intriguing that only MEC cells express C5aR at the protein level

among the bile duct-derived cancer cell lines (Fig. 2B), whereas bile duct carcinomas

showed the highest positive ratio of C5aR expression (Fig. 1B). This fact seems

paradoxical to the result of immunohistochemistry at a glance (Figs. 1, A and B),

implying that C5aR expression was lost during the process of cell line establishment

from primary culture of human cancer cells, as those cells can prioritize expression of

other essential proteins for clonal development in the context of two-dimensional

culture in vitro, which is usually in the absence of C5a.

Cytoskeletal rearrangement and enhanced motility of C5aR-expressing cancer




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cells by C5a stimulus.

In order to analyze the biological effects of C5aR expression in cancer cells under

C5a stimuli, we chose C5aR-negative HuCCT1 cells derived from bile duct carcinomas

of the highest C5aR expression ratio (Fig. 1B). HuCCT1/C5aR cells but not

HuCCT1/mock cells expressed C5aR (Figs. 2, A and B). The cell surface expression of

C5aR in HuCCT1/C5aR cells was confirmed by flow cytometric analysis (Fig. 2C),

which was comparable with that in MEC (Fig. 2C). Since the chemoattractant C5a

causes cytoskeletal rearrangement and stimulates migration of leukocytes (27, 28), we

hypothesized that cancer cells may exploit this mechanism to gain the ability of

migration and invasion by activation of aberrantly expressed C5aR on their cell surface.

To test this, the effect of C5aR activation on actin rearrangement was analyzed by

F-actin immunofluorescence labeling. The majority of cells at the outer edges of

HuCCT1/C5aR cell clusters clearly showed strong filopodia formation 30 min after C5a

treatment (Fig. 3A), which was followed by development of membrane ruffling and

dissolution of stress fibers (Fig. 3A). Three hours after the treatment, such ruffles

disappeared and formation of stress fibers became evident again (Fig. 3A). Some cells at




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the periphery of the cluster even changed their morphology to spindle-like shape and

protruded from the cluster to the vacant area (Fig. 3A). On the other hand,

HuCCT1/mock cells did not show any remarkable changes in both cell morphology and

actin cytoskeleton at any time points after C5a stimulation (Fig. 3A). The time-lapse

video analysis of HuCCT1/C5aR cell movement demonstrated that C5a activated

motility of the cells (Fig. 3B and supplementary Fig. 2 video). Tracing of cell movement

revealed that C5a enhanced motility of HuCCT1/C5aR cells in a dose-dependent

manner, increasing motility 3-fold at 100 nM (Figure 3C), whereas motility of

HuCCT1/mock cells was not significantly affected by C5a (Figs. 3B and 3C). This

experiment confirmed that C5a enhances motility of cancer cells in a C5aR-dependent

fashion.

Enhanced invasiveness of C5aR-expressing cancer cells by C5a stimulus in vitro.

Experiments using Matrigel chambers revealed that C5a stimulated invasion of

HuCCT1/C5aR cells through the matrix layer in a C5a concentration-dependent manner

and enhanced approximately 13-fold over carrier control (PBS) at 10 nM (Fig. 4A). The




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enhancing effect of C5a on invasion of HuCCT1/mock cells was not seen (Fig. 4A).

Intriguingly, cancer cells pretreated with C5a also demonstrated enhanced invasiveness

even in the absence of a C5a concentration gradient (Figs. 4C). This result indicates that

stimulation with C5a during pretreatment is sufficient for enhancing invasion of

C5aR-expressing cancer cells in vitro, and suggests that neither concentration gradient

nor continuous exposure to C5a is required for activating invasion of C5aR-expressing

cancer cells. Similarly, C5a enhanced invasion of MEC cells that endogenously express

C5aR (Fig. 2C), in a C5aR-dependent manner, since this enhancement was abrogated by

a C5aR antagonist (Supplementary Fig. 3) and by the neutralizing antibody against

C5aR, but not by nonspecific IgG (Figs. 4, B and D). Dose-dependent effect of C5a on

invasion was also the case observed in MEC cells (Figs. 4, B and D). To determine

whether C5a-elicited cancer cell migration is dependent on the C5a concentration

gradient, we performed the checkerboard analysis. In addition to C5a

concentration-dependent invasion in the absence of C5a in the upper chamber,

HuCCT1/C5aR cell invasion by 100 nM C5a in the lower chamber was inhibited by

C5a in the upper chamber in a dose-dependent manner (Fig. 4E). Moreover, invasion by




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10 nM C5a in the lower chamber was completely inhibited by 100 nM C5a in the upper

chamber (Fig. 4E). These results suggest that C5a-induced cancer cell invasion is

explained partly by chemotaxis, particularly in the presence of 100 nM C5a. However,

when C5a concentration in the upper chamber was equal to that in the lower chamber,

C5a was still able to induce HuCCT1/C5aR invasion to the significant extent. The

invasion by 10 nM C5a in the lower chamber was not affected by addition of 10 nM

C5a in the upper chamber (Fig. 4E). Furthermore, when 100 nM C5a was added to the

cell suspension in the upper chamber, it triggered significant cancer cell migration even

in the absence of C5a in the lower chamber (Fig. 4E). These results suggest that

enhanced random cell locomotion plays a vital role in the C5a-elicited cancer cell

invasion.

C5a elicits MMP secretion from C5aR-expressing cancer cells.

Degradation of extracellular matrix (ECM) by matrix metalloproteinases (MMPs) is

an essential process for cancer cell invasion (29). The interaction of the chemokine

CXCL12 and its receptor CXCR4 has been shown to increase MMP expression and




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invasion of prostate cancer cells (30). While C5a induces MMP-9 release from human

neutrophils (31), such C5a-elicited MMP release from cancer cells has not been

reported. Interestingly, C5a-enhanced invasion of C5aR expressing cancer cells in the

transwell chambers was significantly hindered by an MMP inhibitor GM6001 (32)

(Figs. 4A and 4B), indicating that enzymatic activity of MMPs plays a crucial role in the

C5a-enhanced invasion of C5aR-expressing cancer cells. Hence we explored the

possibility of C5a provoking MMP secretion from C5aR-expressing cancer cells. MMP

expression array analysis showed that C5a significantly increased release of MMP-1, 3,

9, 10, and 13 from MEC cells, and MMP-8 and 10 from HuCCT1/C5aR cells (Table 1).

These MMPs are known to be associated with both cancer invasion and patient

prognosis (33, 34). Together with inhibition of C5a-enhanced invasion by GM6001

(Figs. 4A and 4B), increased secretion of MMPs by C5a (Table 1) indicates that MMPs

contribute to the C5a-enhanced invasion of C5aR expressing cancer cells. Among

specific MMP inhibitors, an MMP-8 inhibitor exhibited the most significant effect to

impede the MEC cell invasion enhanced by C5 (Supplementary Fig. 3). Intriguingly,

about a 3.2-fold increase in MEC cell invasion induced by C5a at 10 nM (Fig. 4B)




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appeared to correlate with a 3.7-fold increase in MMP-8 (Table 1). These results suggest

that MMP-8 is the most responsible MMP for C5a-elicited MEC cell invasion.

Enhanced invasiveness of C5aR-expressing cancer cells by C5a in vivo.

To evaluate the effect of C5a stimulation on the invasiveness of C5aR expressing

cancer cells in vivo, HuCCT1/mock and HuCCT1/C5aR cells were pretreated with C5a,

labeled with green or orange fluorescent dyes respectively, then mixed to be injected

into nude mice skin. This assay system enables direct comparison of spreading in situ

between two different sublines. This assay revealed that C5a-treated HuCCT1/C5aR

cells spread more broadly than C5a-treated HuCCT1/mock cells in nude mice skin

tissue (Fig. 5A). The most evident induction of the spreading of HuCCT1/C5aR by C5a

was observed at day 2, which was a 1.8-fold increase over HuCCT1/mock (Fig. 5B). In

contrast, there was no difference in spreading between those two sublines of HuCCT1

when they were not treated with C5a (Figs. 5A and 5B). This result suggests that C5a

enhances invasion of C5aR expressing cancer cells in vivo as well as in vitro, and again

stimulation with C5a before injection is sufficient for C5aR expressing cancer cells to




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demonstrate such enhanced invasiveness.

DISCUSSION

C5-derived fragments were reported to enhance cancer cell locomotion, however,

these were not identified as C5a when such reports were made. Rather, they appeared

not to be C5a because of its molecular weight and lack of chemotactic activity for

leukocytes (35, 36). When these studies were performed, the C5aR molecule had not

been identified, and neither recombinant C5a nor C5aR-specific antibodies were

available to prove the activity of C5a to enhance cancer cell migration. In the present

study, we have demonstrated several lines of evidence indicating a crucial role of

C5a-C5aR interaction in cancer cell invasion: [1] C5aR expression was observed in

cancer cells from patients’ tissues and in various human cancer cell lines (Figs. 1 and 2),

[2] recombinant C5a enhanced cancer cell motility (Fig. 3) and invasion both in vitro

(Fig. 4) and in vivo (Fig. 5), and [3] enhanced cancer cell invasiveness elicited by the

C5a-C5aR axis was dependent on the increased release of MMPs (Fig. 4 and Table 1),

proteases that are indispensable for cancer cell invasion towards surrounding tissues




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(29). C5aR expression is essential for cancer cells of epithelial origin to enhance

motility and invasiveness by C5a, given that C5aR expression is required for any

remarkable changes in cell morphology (Fig. 3A) and enhanced invasiveness (Figs. 4A

and 4C) in HuCCT1 cells after C5a stimulation (Figs. 5A and 5B). In addition, a C5aR

antagonist (Supplementary Fig. 3) and by a neutralizing antibody against C5aR (Figs. 4,

B and D) abrogated C5a enhanced invasion of MEC cells. These data are consistent

with the phenomenon that C5a enhances cancer cell invasion via C5aR, and to our

knowledge, this is the first report that shows the biological role of the C5a-C5aR axis in

human cancer cell invasion.

C5aR being expressed in cancerous cells but not normal epithelial cells except

kidney proximal tubular epithelial cells in human tissue specimens (Fig. 1A, and

supplementary Fig. 1A) may indicate C5aR expression to be a consequence of

malignant transformation. A similar example is CXCR4: the receptor of CXCL12 that is

a potent chemoattractant like C5a and is produced in the cancer microenvironment (20).

This receptor is commonly found in cancer cells and its expression is induced by factors

such as hypoxia, vascular endothelial growth factor and estrogen in the cancer




- 28 -

microenvironment. CXCR4 expression is also activated by mutations in genes that alter

levels of hypoxia-inducible factors, and gene fusion events. IL-6-induced C5aR

expression in rat hepatocytes (37) suggests that C5aR can be expressed in response to

specific cytokines that are rich in the cancer microenvironment (20, 38). However, IL-6

and IFN-γ did not induce C5aR expression in HuCCT1 cells (unpublished data). This

may suggest that C5aR expression is dependent on genetic events characteristic in

individual cancers. Such differences might reflect variation in C5aR-positivity in

different primary organs (Fig. 1B).

Leaky cancer vasculature facilitates the supply of the complement system

components from the bloodstream to cancer tissues (39), where as shown in an animal

cancer model (2), C5a is generated through activation of the complement system in

response to cancer cells (3), although they are protected from complement attack by

complement regulators (6). Indeed, C5a is detectable in human plasma incubated with

MEC or HuCCT1 cells in vitro (Supplementary Fig. 4). Besides this pathway, C5a is

possibly generated directly from C5 through thrombin-dependent cleavage (12),

following the coagulation reaction initiated by tissue factor that can be expressed on




- 29 -

cells in cancer tissues including cancer cells, fibroblasts and activated leukocytes (13).

C5a can also be generated from C5 by a serine protease from activated phagocytes (14)

recruited to the cancer tissue. In addition, compared with tightly adhering

non-cancerous epithelial cells, the loose cell-to-cell contact of cancer cells allows the

generated C5a to access the cancer cell membrane, enabling it to bind to C5aR. Thus,

C5a produced in the cancer microenvironment can be predicted to activate

C5aR-positive cancer cells to promote migration from the primary site.

C5a induced dynamic sequential reorganization of actin cytoskeleton in

C5aR-expressing cancer cells, namely, filopodia formation, membrane ruffling then

formation of stress fibers (Fig. 4A). These processes have been reported to be provoked

by activation of Cdc42, Rac1 and RhoA, respectively (40). Such sequential activation of

these small G proteins, which is a robust driving force for cell movement, has been

documented previously (41). In fact, C5a induces activation of Cdc42 and Rac1 in

neutrophils, leading to actin reorganization of the cell (42). We are currently studying if

the C5a-C5aR axis can activate upstream signaling pathways of those small G proteins

in cancer cells.




- 30 -

Together with motility stimulation in the C5a-containing culture medium (Fig. 3),

increased invasiveness of C5a-treated C5aR-positive cancer cells in the matrix gel (Fig.

4) and in nude mouse skin (Fig. 5) in the absence of a C5a concentration gradient

suggest that C5a enhances cancer cell random locomotion instead of inducing

chemotaxis, which is supported by the checkerboard analysis (Fig. 4E). If C5a were

only chemotactic for cancer cells, stimulated cells would be expected to assemble in the

primary cancer site where C5a is released and enriched; thus C5a would hinder cancer

cell spread. In contrast, enhanced random cell locomotion would be more relevant for

promoting cancer cell invasion and spreading, namely, the phenomenon that cells leave

away from the source of the stimulant. Accordingly, such C5a activity is presumed to

favor cancer cell dissemination from the primary site (Fig. 5).

This study implies that the C5a-C5aR axis could be a target for anti-cancer therapy.

For instance, depleting C5 with anti-C5 antibodies would suppress C5a generation in

cancer tissues. Therefore this suggests that C5aR may also become a possible and

feasible target for molecular-based medicine by generating specific antagonists. Such

agents may provide useful therapeutic options for cancer treatment in the future.




- 31 -

Acknowledgments

We thank Ms. T. Kubo for technical assistance with the immunohistochemical staining,

and Mr. K. Matsuda, KEYENCE, for the fluorescence microscope imaging, and

analysis. This work was supported in part by a Grant-in-Aid for Scientific Research (C)

from the Japanese Ministry of Education and Science (Grant No. 22590363 to T.I.).




- 32 -

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Figure legends

Figure 1. Expression of C5aR in human primary cancers. A, Human cancer tissues were

immunohistochemically stained with an anti-C5aR antibody or control IgG (inset), and

representative examples are shown. Colon cancer (top left) and normal colon epithelium

(top right); esophagus normal epithelium (bottom, encircled) and cancer (bottom, not

encircled). Scale bars = 50 µm. B, Cancer cell C5aR-positive case ratios (positive

cases/total cases examined) () in primary organs. The vertical scale bar shows the 95%

confidence interval that is estimated from the binominal proportion based on the beta

distribution. EP, esophagus (n = 20); ST, stomach (n = 20); Col, colon (n = 40); Liv,

liver (n = 20); BD, bile ducts (n = 46); Pan, pancreas (n = 10); Kid, kidney (n = 11); UB,

urinary bladder (n = 45); Lun, lung (n = 12); PT, prostate (n = 25); Mam, mammary

gland (n = 21).

Figure 2. Expression of C5aR on cancer cell lines including HuCCT1/C5aR and

HuCCT1/mock. A, Expression of C5aR mRNA in several cancer cell lines shown by

RT-PCR. B, Expression of C5aR protein in bile duct and colon cancer cell lines, shown




- 40 -

by immunoblotting using an anti-C5aR antibody. β-actin mRNA and protein were used

as controls. C, Expression of C5aR on the cell membrane in MEC and HuCCT1/C5aR,

shown by flow cytometry using FITC-conjugated anti-C5aR antibody (gray line) or

control antibody (black line).

Figure 3. C5a elicits C5aR expressing cancer cells by inducing cytoskeletal

reorganization and changes in cellular morphology. A, HuCCT1/C5aR and

HuCCT1/mock cells were incubated with C5a (100 nM) and fixed at indicated time

points. F-actin was visualized by immunofluorescence staining with Alexa

488-conjugated Phalloidin. Scale bars: 20 µm. Arrow head and an arrow indicate

filopodia and membrane ruffling, respectively. B, Time Lapse analysis of cell motility.

Cell images taken at 0, 3, 6 and 9 h are shown. Broken circles indicate the initial cell

position shown at 0 h. C, Cell migration distance was measured by tracing a cell. ＊P <

0.01 (n = 6).

Figure 4. Enhancement of C5aR-expressing cancer cell invasion by C5a in vitro.




- 41 -

Cancer cell invasion was measured by the Matrigel invasion chamber assay.

HuCCT1-derived (A) or MEC cells (B) were placed in the upper chamber and medium

supplemented with various concentrations of C5a was placed in the lower chamber.

HuCCT1-derived (C) or MEC cells (D) were pretreated with various concentrations of

C5a for 12 h and 24 h respectively. These cells were washed and were placed in the

upper chamber, and medium was placed in the lower chamber. Incubation time for

assay: 24 h (A, C) and 36 h (B, D). (□), HuCCT1/mock (A, C); (■), HuCCT1/C5aR (A,

C); crosshatched and closed bars (B, D) indicate MEC cells treated with anti-C5aR

antibody or nonspecific IgG, respectively. Gray bars (A, B) indicates cells incubated in

the upper chamber in the presence of GM6001 (5 µM). E. Checkerboard analysis for

C5a cancer cell invasion activity. Closed, crosshatched and open bars indicate C5a

concentration 0, 10 or 100 nM, respectively, in the lower chamber.＊, p < 0.01; n.s., not

significant.

Figure 5. Enhanced invasiveness of C5aR-expressing cancer cells by C5a in vivo.

HuCCT1/C5aR and HuCCT1/mock cells were incubated separately in the presence or




- 42 -

absence of 100 nM C5a (C5a(+) or C5a(-), respectively) for 12 h. After washing, cells

were labeled, mixed, and injected together into nude mouse skin. A. Distribution of

HuCCT1/C5aR (orange) and HuCCT1/mock (green) cells in nude mouse skin two days

after implantation. Right panels are corresponding sections stained with

hematoxylin-eosin. Bars = 300 µm. B. Cell distribution square ratio (HuCCT1/C5aR

cells versus HuCCT1/mock cells). Cells were incubated in the presence (■) or absence

(□) of C5a. ＊, p < 0.01 (n = 5).




esophagus

colon

A

B

0

20

40

60

80

100

po

sitiv

ity (

%)

EP

ST

Col

Liv

BD

Pa

n

Kid

UB

PT

Lun

Ma

m

Figure 1




A

B

C

C5aR(+)HuCCT1HuCCT1/C5aR

100 101 102 103 104

200

160

120

80

40

0

ce

ll counts

C5aR expression

HuCCT1/mock

100 101 102 103 104

200

160

120

80

40

0

ce

ll counts

C5aR expression

MEC

100 101 102 103 104

200

160

120

80

40

0

cell

coun

ts

C5aR expression

C5aR

β-actin

ME

C

Hu

CC

T1

SS

p-2

5

RB

E

mo

ck

C5aR

DL

D1

SW

62

0

HC

T1

5

CO

LO

20

5

HC

T1

16

bile duct colon

HuCCT1

C5aR

β-actin

ME

C

bile duct colon

Hu

CC

T1

SS

p-2

5

RB

E

mo

ck

DL

D1

SW

62

0

HC

T1

5

CO

LO

205

HC

T1

16

HuCCT1

C5aR

Figure 2




HuCCT/C5aR

HuCCT/mock

0 min 30 min 60 min 180 min 360 min

A

C5a Concentration (nM)

ce

ll m

igra

tion (

µm

)

200

400

600

800

1000

0 10 100

≈≈

≈≈

*

n.s.

*

B

C

HuCCT1/C5aR

HuCCT1/mock

C5a(100 nM)

(+)

(+)

( )

( )

0 3 6 9

Time Lapse (hr)

Figure 3




0

5

10

15

0 1 10 100

*

*

**

C5a concentration (nM)

mig

rate

d c

ell

ratio (

fold

)

0

5

10

0 1 10 100

*

*

*


mig

rate

d c

ell

ratio (

fold

)

A B

C Dm

igra

ted c

ell

ratio (

fold

)

0

1

2

3

4

5

0 1 10 100

*

*

*

*

n.s.


mig

rate

d c

ell

ratio (

fold

)

0

1

2

3

0 1 10 100

*

* *

n.s.

Figure 4

E

0

5

10

15

mig

rate

d c

ell

ratio (

fold

)

0 10 100C5a in the upper chamber (nM)

** *

**

n.s.

n.s.


+ G

M

+ G

M

+ a

nti-

C5

aR

+ a

nti-

C5

aR

+ n

on

sp

ecific

IgG

+ n

on

sp

ecific

IgG




C5aR

(+) ( - )merge

C5a

(+)

( - )

A

B

1

2

Ce

ll D

istr

ibu

tion A

rea

Ratio

C5a

R(+

)/C

5aR

(-)

(fold

)

1 2 3 (day)

*

*

Figure 5




Table 1. C5a-stimulated MMP release from C5aR expressing cancer cells.

MEC HuCCT1/C5aR

MMP C5a ( - ) a C5a ( + ) a Ratiob C5a ( - ) a C5a ( + ) a Ratiob

1 149332.6±17039 291140.4±5360.4* 1.95 88906.6±27436.8 129408±3485 1.46

2 32.5±11.3 116.9±67. 8 3.59 1220.4±184.3 870.7±73 0.71

3 26.8±12.0 134.7±42.1* 5.03 971.6±224.6 927.5±162.9 0.95

8 20.1±23.6 74.9±29.9 3.72 335.6±77.7 1128.3±120.9* 3.36

9 < 0.1c 12.3±3.6* - 6105.1±2011.3 6213.4±4357.7 1.02

10 857.6±740.5 9301.4±641.8* 10.84 57641.4±5197.5 111666.4±7911.1* 1.94

13 359.2±118.7 1367.3±309.3* 3.80 1411.9±154.6 1642.8±287 1.16

a MMP concentrations (pg/mL) in culture supernatants of cancer cells cultured for 24 h in

the presence (+) or absence (-) of C5a (100 nM). b ratio of C5a(+) versus C5a(-) in mean

values. c below the detection limit. *P < 0.01 (n = 4).




Published OnlineFirst January 3, 2013.Clin Cancer Res Hidetoshi Nitta, Yoshihiro Wada, Yoshiaki Kawano, et al. (CD88).anaphylatoxin C5a via aberrantly expressed C5a-receptor Enhancement of human cancer cell motility and invasiveness by

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