Adenoid Cystic Carcinoma

Adenoid Cystic Carcinoma

Oral Science International, November 2005, p.69-79 Copyright © 2005, Japanese Stomatology Society. All Rights Reserved. Review Article Adenoid Cysti...

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Oral Science International, November 2005, p.69-79 Copyright © 2005, Japanese Stomatology Society. All Rights Reserved.

Review Article

Adenoid Cystic Carcinoma : Participation of Urokinase-type Plasminogen Activator (uPA)and its Receptor(uPAR)in Tumor Invasion Kanemitsu Shirasuna Department of Oral and Maxillofacial Surgery, Graduate School of Dental Science, Kyushu University (Chief : Kanemitsu Shirasuna) Abstract : Adenoid cystic carcinoma(AdCC)is one of the most common malignant tumors of the salivary glands and has unique clinical features and behavior. AdCC grows slowly, but spreads relentlessly into adjacent tissues, with a proclivity for invading nerve and endothelial sheaths. Moreover, the frequency of recurrence and distant metastasis of AdCC is very high. In vivo and in vitro, AdCC produces a large amount of extracellular matrix(ECM) , including basement membrane(BM)components, elastin, and mucopolysaccharides. The accumulation of ECM components in intercellular spaces results in the formation of a pseudocyst, which is the characteristic architecture of AdCC. AdCC cells degrade considerable amounts of mesenchymal-elaborated ECM through the urokinase-type plasminogen activator(uPA)-plasmin system. By contrast, tumor-produced ECM is resistant to degradation, because it contains plasminogen activator inhibitor type 1 (PAI-1) . The migration response of AdCC cell lines to ECM, especially type I and type IV collagens, is much stronger than that of oral squamous cell carcinoma(SCC)cell lines, while both cell types generally show similar patterns of integrin subunit expression. The AdCC cell response to collagens is largely and exclusively inhibited by anti-α2 integrin antibody. Surface uPA receptor(uPAR)expression by AdCC cell lines is greater than that by SCC cell lines and increases in response to collagen stimulation. This is accompanied by the assembly of numerous focal adhesions, consisting of the adapter proteins uPAR,α2 integrin, vinculin, and paxillin. A role for uPAR in cell migration and assembly of adaptor proteins was also demonstrated by transfecting AdCC cells with an antisense uPAR RNA, which strongly reduced both responses. Therefore, the proclivity of AdCC cells to migrate to type I and IV collagens might be due to the overexpression of uPAR, which also plays a key role in focal adhesion assembly. In conclusion, the invasiveness of AdCC cells might be regulated by the interaction of uPA-uPAR with integrin. Key words : adenoid cystic carcinoma, recurrence, invasion and metastasis, urokinase-type plasminogen activator(uPA), uPA receptor, plasminogen activator inhibitor type 1, matrix degradation, integrins, cell migration, focal adhesion

1. Introduction  Received 10/7/05 ; accepted 11/1/05.  Grant support : Ministry of Education, Science, Sports and Culture of Japan ; No. 16390593.  Requests for reprints : Kanemitsu Shirasuna, Department of Oral and Maxillofacial Surgery, Graduate School of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 8128582, Japan, Phone : +81-92-6426388, Fax : +81-92-6426392, E-mail : [email protected]

 Adenoid cystic carcinoma(AdCC)is one of the most common malignant tumors of the salivary glands. The tumor grows slowly, but spreads relentlessly into adjacent tissues, so recurrence is common and distant metastasis, especially to the lungs, is very frequent1. Since AdCC is resistant to radiotherapy and chemotherapy,

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effective forms of treatment are required. Histological-

-anchored uPAR, which consists of three homologous do-

ly, the tumor is characterized by a cylindrical or cystic

mains(D1, D2, and D3). The binding of proteins such

stroma surrounded by anastomosing cords of epithelial

as uPA, vitronectin, and PAI-1 to uPAR involves a direct

tumor cells. The cystic stroma and the interstitium sur-

interaction with D1, but requires the integrity of the

rounding the tumor islands are often hyalinized and

full-length receptor7. uPA bound to uPAR exhibits en-

contain various substances, including collagen-like fi-

hanced proteolytic activity and directly activates plas-

bers, elastin, basement membrane constituents, and

minogen and matrix metalloproteases, which, in turn,

1

mucopolysaccharides .

enhance ECM degradation7. The receptor-binding do-

 Invasion and metastasis are characteristic features of

main of uPA is located in the amino-terminal fragment

malignant tumors. Although the molecular mechanisms

(ATF ; residues 1-135)of the uPA molecule and does

underlying metastasis are complex, it is clear that tu-

not involve the protease domain.

Independent of its

mor cells must migrate through the extracellular matrix

proteolytic function in matrix degradation, the uPA-

(ECM)in order to invade local tissues and metastasize

uPAR interaction 1)mediates several signaling events,

to distal sites. Migrating cells use both adhesion mole-

2)regulates cell adhesion on vitronectin-coated surfac-

cules and proteolytic enzymes to regulate their interac-

es, 3)modulates integrin activity, 4)triggers cell mi-

tion with and response to the ECM, and cooperation be-

gration, and is strongly correlated with the metastatic

tween these components operates at several levels :

potential of various tumors10.

integrin signaling induces proteases2, proteases co-local-

 Integrins are ubiquitous, heterodimeric transmem-

ize with integrins3, and proteases regulate the interface

brane receptors that anchor the cell to the ECM and the

4

A protease

cytoskeleton to the plasma membrane11. Integrin recep-

system intimately connected to integrins comprises uro-

tors mediate cell adhesion, migration, and bidirectional

kinase-type plasminogen activator(uPA) , the uPA re-

signal transduction via interactions with ECM pro-

ceptor(uPAR) , and plasmin5.

teins11,12. In particular, the ECM-integrin interaction

 The plasminogen activation system, which is made up

leads to the reorganization of the actin cytoskeleton,

of plasminogen activators, their inhibitors, plasmino-

initiates signal transduction cascades, and coordinates

gen, and the respective cell-surface-binding proteins

the response to growth factors12. Recent work points to

and receptors, plays a central role in cell invasion.

important interactions between integrins, cytoskeletal

Along with tissue-type plasminogen activator, uPA pro-

components, and signaling molecules13.

motes the formation of plasmin, the key protease in fi-

 Focal adhesions are specialized cell adhesion sites

brinolysis. Plasmin activates matrix-metalloproteases,

that transduce signals from the ECM intracellularly.

which constitute a proteolytic system for cell migration

The assembly and disassembly of focal adhesions result

between integrins and the cytoskeleton .

6, 7

and tissue remodeling . Together, these proteases fa-

in the formation and displacement of cellular stress fi-

cilitate cell invasion by(1)activating latent growth

bers. On ligand activation, transmembrane ECM recep-

factors or releasing them from their ECM-binding sites,

tors, such as integrins, cluster at focal adhesions, which

and(2)degrading a variety of proteins in the ECM.

are physically connected to the cytoskeleton by stable

Different plasminogen activator inhibitors(PAIs)neu-

and transient interactions14. A characteristic property

tralize plasminogen activator, primarily fast-acting

of most focal adhesion proteins is their multi-domain

8

PAI-1 . PAI-1 is a serine protease inhibitor, but, unlike

structure. For example, vinculin can interact with at

other proteases, its active conformation is stabilized by

least ten other focal adhesion proteins, including actin,

high-affinity binding to ECM-associated vitronectin. In

tensin, paxillin, and talin, although probably not simul-

addition to producing uPA, malignant cells also synthe-

taneously15. In addition to recruiting non-catalytic com-

size the uPA inhibitor PAI-1, thereby regulating PA ac-

ponents, protein kinases also cluster at focal adhesions

tivity levels in the cellular microenvironment8,9. There-

in response to cell adhesion, which leads to increased

fore, tumor-cell-mediated proteolysis may be controlled

phosphorylation of focal adhesion proteins15. Paxillin,

by the fine regulation of tumor-cell-secreted proteases

another important multi-domain adaptor protein, func-

and protease inhibitors.

tions in recruiting both signaling and structural mole-

 uPA binds to a glycosyl phosphatidylinositol(GPI)

cules to focal adhesions16. The COOH terminus of paxil-

November, 2005

Adenoid Cystic Carcinoma

71

lin is composed of four Lin 11, Isl 1, Mcc 3(LIM)

lung, followed by other organs, such as bone and liver.

domains, which mediate paxillin targeting to focal adhe-

Perineural invasion is one parameter predicting distant

sions17.

metastasis1,21.

The NH2 terminus of paxillin supports the

binding of vinculin and several protein tyrosine kinases,

 Unlike most other types of salivary gland carcinoma,

including focal adhesion kinase(FAK), proline-rich ty-

in AdCC distant metastasis is far more frequent than

rosine kinase 2(PYK2), C-terminal Src kinase(Csk) ,

regional lymph node involvement, and this is why elec-

and Src. Paxillin leucine-rich sequences(LDXLLXXL),

tive neck dissection is not indicated. Radiation therapy

called LD motifs, serve as binding sites for both FAK

may improve local tumor control, especially when there

and vinculin17.

is microscopic residual tumor22-24. Fast neutron radio-

 AdCCs are characterized by their relentless spread

therapy provides higher local control rates than conven-

into adjacent tissues, and their high frequency of recur-

tional photon radiation therapy, even for palliation of

rence and distant metastasis. Tumor invasion is the

unresectable or recurrent tumor25,26. While the 5-year

first critical step in a complex process that leads to the

survival rate of patients with AdCC is relatively favor-

formation of metastases, and the mechanism behind

able, because of the invasiveness of the tumor and its

this step is the focus of this review.

resistance to conventional radiotherapy and chemotherapy, the long-term prognosis is extremely poor in most

2. Salient clinical features related to tumor inva-

patients. Therefore, the development of effective new

sion

approaches for treating AdCC is urgently needed.

 AdCC occurs most often in adults in the fifth through seventh decades of life. While a female predominance

3. Histological characteristics

for AdCC has been reported in some studies18, others

 AdCC is composed of distinct populations of cells,

did not find gender-specific differences in the incidence

which can be divided into two types based on ultrastruc-

of these tumors16,19. AdCC occurs predominantly in the

tural and histochemical studies1,27-29. Type A cells com-

submandibular, parotid, and minor salivary glands,

prise cells of the pseudocyst lining, nonluminal cells,

with more than half of the tumors in the latter located

and peripheral cells, and exhibit several myoepithelial

in the palate. The tumor is apparent in the major or

markers. Type B ductal or luminal cells express several

minor salivary glands as a swelling or mass. It grows

secretory markers. While type A cells are abundant in

slowly, but spreads relentlessly into adjacent tissues.

tumor tissue, type B cells are rather scarce. Immuno-

Invasive AdCC of the parotid gland leads to paralysis of

histochemistry studies have shown that type A cells are

the facial nerve, whereas pain and ulceration often de-

positive for muscle-specific actin, low-molecular-weight

velop in tumors of the minor salivary gland.

cytokeratin, and S-100, whereas type B cells express

 It has generally been accepted that surgery is the

carcinoembryonic antigen(CEA) , epithelial membrane

preferred treatment for AdCC, and complete excision at

antigen(EMA) , high-molecular-weight cytokeratin27.

initial surgery is associated with a favorable survival

Co-expression of cytokeratin and vimentin has been ob-

rate and cure. Nonetheless, in about 40% of patients

served in tumor cells of a variety of salivary gland neo-

there is recurrence of the tumor after surgery. Since

plasms, including AdCC, pleomorphic adenoma, and

margin status significantly influences local control of

carcinomas in pleomorphic adenoma30,31. By contrast,

the tumor and ultimately patient survival, careful his-

human normal salivary glands co-express these pro-

tological observation of the surgical margins is essen-

teins, but only in areas where myoepithelial cells and

tial. AdCCs tend to proliferate discontinuously, with

basal cells of the epithelium are present. Nonhomoge-

tiny scattered tumor nests that spread widely and deep-

neous expression of intermediate filaments in AdCC has

35

ly into adjacent tissues .

Cumulative evidence sug-

also been shown immunohistochemically. Cytokeratin

gests that microscopic deposits to distant sites occur

was more striking in luminal cells(type B)of the tu-

early in the growth of the primary tumor. Indeed, one

mor than in peripheral cells, indicating an inverse cor-

third to half of AdCC patients develop metastases dur-

relation between cytokeratin and vimentin expression

ing the follow-up period, even if the primary tumor was

in AdCC27,30,32. This observation also suggests a gradu-

20

controlled . The most frequent site of metastasis is the

al transition from basal to luminal cells, equivalent to

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the differentiation seen in normal stratified epithelium.

4. Biological characteristics of cultured cells

 AdCC cells can also be divided into three subgroups histologically : cribriform, tubular, and solid(Fig. 1) .

1)ECM production and degradation by AdCC-de-

The most characteristic pattern is cribriform, created by

rived cell lines in vitro are regulated by the uPA-

many pseudocysts scattered in cell islands(Fig. 1C) .

plasmin system.

29

Perzin et al. reported that patients whose tumors con-

 Well-characterized cell lines established from human

sisted of the tubular or cribriform pattern had a favor-

AdCCs, such as Acc-2, Acc-337, and Acc-M38(established

able outcome, in contrast to patients with solid type.

at Shanghai Second Medical University), and ACCS9,39,

Several reports have indicated that the cribriform pat-

ACCY32, and ACCT,

tern is associated with the longest patient survival

standing the biology of this type of tumor. AdCCs vigor-

time, followed by tubular and solid types1,21,22. Howev-

ously produce and accumulate ECM, including base-

er, other studies were unable to find a relationship be-

ment membrane components. As it does in vivo, the

tween histological pattern and survival, since individual

ACCY, ACCS, Acc-2, and Acc-3 cell lines have been

33

serve as useful tools for under-

tumors often contained all three cell types . Perineural

shown to produce large amounts of ECM, including col-

invasion may be more associated with an unfavorable

lagen, elastin, basal lamina components, and mucopoly-

outcome(Fig. 1D).

saccharides1,32,34,40. The amount of ECM produced by

 ECM-containing BM components are a histological

ACCS cells is comparable to that produced by normal

feature of AdCC. They are observed in the cribriform

mesenchymal cells. ACCS and ACCY cells are of the

and tubular patterns of the pseudocyst lining and on

basal-myoepithelial cell type(see Sect. 2, type A cells)

the outer surface of cells comprising tubular areas. BM

and form pseudocysts in vitro. This finding supports

consists of several types of macromolecules, including

suggestions that myoepithelial-like cells are responsible

type IV collagen, laminin, fibronectin, heparin sulfate

for ECM production, including basal lamina macromole-

proteoglycan, and entactin. These proteins have been

cules, and that the accumulation of ECM in intercellu-

demonstrated in the ECM surrounding AdCCs, in the

lar spaces contributes to the formation of the stroma

pseudocystic spaces, hyaline stroma, and around tumor

surrounding AdCCs32.

nests34.

Tumor cells appear to proliferate in contact

 In addition to extensive ECM-producing activity,

with the BM and to infiltrate through BM-rich tissues

ACCS cells can degrade considerable amounts of ECM

(Fig. 2), such as peripheral nerves, blood vessels, and

elaborated by mesenchymal cells, mainly via the uPA-

skeletal muscle35.

It has been clearly demonstrated

plasmin system9. ACCS cells produce high levels of sev-

that BM molecules in the stroma are produced by the

eral proteolytic enzymes, including uPA, MMP-2, and

tumor cells themselves32(see sect. 4). Furthermore,

MMP-9 and ECM degradation by the cells is both plas-

the biosynthetic and metabolic changes that occur in

minogen-dependent and closely associated with uPA ex-

the ECM might be involved in malignant transforma-

pression9. In vitro study has demonstrated that ECM

tion.

produced by ACCS cells contains PAI-1(Fig. 1E)and

 Many histochemical- and morphological-based obser-

is consequently resistant to degradation9. As a result,

vations suggest that myoepithelial-like cells are respon-

ECM components can accumulate in the intercellular

sible for the production of ECM components, including

spaces, thereby forming a pseudocyst(Fig. 1C and Fig.

32

BM molecules . We previously found a correlation be-

2), which is the characteristic architecture of AdCC32.

tween an increase in glycosaminoglycan synthesis and

Since, in this way, AdCC cells are surrounded by their

the expression of a myoepithelial-like phenotype in cul-

own ECM, as well as by normal host connective tissue,

tured cells36. The production of ECM and BM by AdCC

they are able to proliferate along the tumor-produced

cells is exclusively a myoepithelial or basal cell charac-

ECM and then bind to and finally invade adjacent nor-

teristic, and is vigorous, but disorganized, compared

mal tissues(Fig. 2).

with production in normal tissue.

November, 2005

Adenoid Cystic Carcinoma

73

2)EGF induces activation of AP-1, which simu-

SCC cells have very similar profiles of integrin subunit

lates ECM-degrading protease production and

expression51. The collagen-stimulated migration of AdCC

migration, while dexamethasone inhibits these

cells is largely and exclusively inhibited by anti-α2 inte-

events

grin antibodies, suggesting that α2 integrin is function-

 Certain growth factors, such as epidermal growth fac-

ally important for the adhesion and migration of AdCC

tor(EGF), regulate tumor invasiveness by altering cell

cells to collagen.

adhesion, ECM-degrading protease production, and motility41,42. Treatment of AdCC cells and oral squamous

4)AdCC cell migration to collagens may be due

cell carcinoma(SCC)cells with EGF stimulates sever-

to uPAR overexpression, which plays a key role in

al activities, such as ECM degradation, migration, and

focal-adhesion assembly and migration

Matrigel(artificial basement membrane protein)inva-

 Various integrin-associated proteins have been re-

9, 43, 44

. In various types of cells, including AdCC and

ported to modulate integrin signaling and to control

SCC cells, EGF induces increased expression of uPA

integrin-mediated events. The multifunctional surface

and uPAR proteins and mRNA through activation of

receptor uPAR is one such protein that controls signal-

44 . Both transcription factor activator protein-1(AP-1)

ing, adhesion, and migration52. Compared with normal

EGF-induced invasion and uPA and uPAR expression

cells, malignant tumor cells usually express higher lev-

are markedly inhibited by dexamethasone(DEX)

els of uPA and uPAR5,53,54, which results in increased

through a reduction of DNA-AP-1 binding and the in-

tumor cell invasion in vitro44. The results of our studies

44

report-

showed that AdCC cells produce a large amount of uPA

ed that EGF also stimulates the production of MMP-1,

and degrade ECM via the uPA-plasmin system9, and

stromelysin-1, and MMP-9 in a process that may be

that uPAR surface protein and mRNA expression are

mediated by AP-1.

MMPs, particularly MMP-2 and

greater in AdCC than in SCC cell lines39. Combined,

MMP-9, are important for the degradation and invasion

these findings suggest that the characteristic properties

sion

42

duction of PAI-1 expression . Rosenthal et al.

45, 46

Initially, MMPs released from

of AdCC cells, invasion and metastasis, are mainly reg-

cells are inactive ; they are converted to active forms by

ulated by the uPA-uPAR system. In this respect, uPAR

of BM and ECM

.

47, 48

. Since uPA is activated by

has been shown to facilitate cell-surface plasminogen

binding with uPAR, the uPA-uPAR interaction is essen-

activation, which generates a proteolytic cascade con-

tial for augmenting proteolytic activity and uPAR-medi-

tributing to matrix degradation during tumor invasion,

ated signaling, which ultimately induce motility and in-

and might function in cell adhesion and migration9,44.

vasion. DEX inhibits the expression of both uPA and

Several studies have demonstrated the presence of

uPAR by blocking AP-1 activation, and therefore may be

uPAR at focal adhesion sites and at leading edges of mi-

other MMPs or plasmin

44

useful in the treatment of malignant tumors .

grating cells, suggesting that uPAR physically associates with integrins55-57. Figure 4B shows a cell clone

3)AdCC migrates to types I and IV collagens

transfected with pECF-N1 vector containing uPAR

 As noted above, due to its proclivity for invading

cDNA and cDNA for the enhanced cyan fluorescent vari-

1, 49

nerve and endothelial sheaths

, the frequency of re-

ant of green fluorescent protein(CFP).

CFP-uPAR

currence and distant metastasis of AdCC is very high.

overexpression resulted in an increased frequency of the

Data from our laboratory suggest that AdCC cells ini-

formation of lamellipodia, in which CFP-uPAR was con-

tially proliferate along tumor-produced ECM, contain-

centrated58. This finding indicates that receptor expres-

ing BM components, and that these invasive cells ad-

sion plays a role in actin reorganization, which is regu-

here and migrate to nerve and endothelial sheaths,

lated by genes of the Rho family. When AdCC cells were

which also contain BM components(Fig. 2). In fact,

plated on collagen, the surface level of uPAR increased,

ACCS-produced ECM strongly stimulates the migration

and numerous focal adhesions, consisting of uPAR, vin-

50

of these cells in vitro . Moreover, the ability of ACCS

culin, and paxillin, were assembled(Fig. 4A, C and D) ,

and ACCT cells to migrate and adhere to ECM, espe-

whereas collagen-stimulated SCC cells or AdCC cells

cially types I and IV collagen, is greater than that of

plated on other types of ECM, such as fibronectin, failed

oral SCC cell lines(Fig. 3)39,51, although AdCC and

to assemble focal adhesions39. Figure 4C shows a case

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Oral Science International Vol. 2, No. 2

Fig. 1

Fig. 2

Fig. 3

Fig. 4

November, 2005

75

Adenoid Cystic Carcinoma

of comparison of a transfectant of ACCS with uPAR an-

integrin, paxillin, and vinculin at focal adhesions

tisences(ACCS-AS)and control clone(ACCS-zeo) .

following collagen(Fig. 4C and D) , but not fibronectin,

The surface uPAR level of AdCC cells was up-regulated

stimulation39. These findings suggest that on collagen

by collagen stimulation in the absence of uPAR mRNA

activation, uPAR migrates to the focal adhesion via its

induction39. As shown in experiments with EGF, uPAR

interaction with a collagen receptor, probably the α2 in-

expression is controlled mainly at the transcriptional

tegrin receptor.

level44. Post-transcriptional regulation and recycling of

which is a GPI-anchored protein, plays an important

uPAR to the plasma membrane represent additional

role in regulating cell migration via the modification of

levels of regulation7. In a study applying immunocyto-

actin-integrin adhesive complexes6,55,57,59,60.

chemical staining, we demonstrated the translocation of

 The down-regulation of uPAR inhibits the growth and

uPAR from the cytoplasm to focal adhesions on collagen

migration of carcinoma cells in vitro61,62, alters cytoskel-

stimulation of AdCC cells, while SCC cells expressing

etal organization in human glioma cells63 and fibro-

low levels of uPAR were less able to assemble focal

blasts64, and inhibits colon cancer metastasis in a nude

adhesions. In AdCC cells, uPAR co-localized with α2β1

mouse model65. However, how down-regulation of uPAR

Recent reports indicated that uPAR,

Fig. 1

Adenoid cystic carcinoma(AdCC)is composed of distinct populations of cells that can be arranged in three main patterns : solid(A) , tubular(B) , and cribriform(C) . The most characteristic pattern, cribriform, is created by many pseudocysts containing extracellular matrix(ECM)produced by AdCC cells. Perineural infiltration is also a characteristic feature of this tumor(D) . E, Analysis of radio-labeled ECM of cultured AdCC cells and fibroblasts by SDS-PAGE and autoradiography. Whole and IP(PAI-1)indicate whole ECM and immunoprecipitation of the ECM with anti-PAI-1 antibody, respectively. The intensity of PAI-1 band(MW 45 kDa)in AdCC ECM(lane 2)is greater than that of fibroblast ECM(lane 4) .

Fig. 2

Invasion by adenoid cystic carcinoma(AdCC)cells is regulated mainly by urokinase-type plasminogen activator(uPA) and uPA receptor(uPAR). AdCC cells initially grow as a solid mass[1]and produce a large amount of extracellular matrix(ECM), which accumulates in the intercellular space, resulting in the formation of a cribriform pattern, created by many pseudocysts. Since patients with the cribriform pattern have a favorable outcome, pseudocyst forming cells might be less invasive. After the loss of intercellular junctions, for example through reduced expression or cleavage of cadherins, invasive tumor cells detach[2]and then migrate preferentially into tumor-produced or mesenchymal produced-ECMs, collagens[3] , via interaction of integrin, protease(uPA)and protease-receptor(uPAR) . The high affinity of invasive cells for type IV collagen allows their further migration into nerve sheaths, resulting in paralysis of the facial nerve and pain, and into vascular endothelial sheaths[4], resulting in distant metastasis. After AdCC cells intravasate into the bloodstream[5], the tumor cells, which have platelet aggregation activity[6], adhere to vascular endothelial cells of distant organs and extravasate into those tissues. Subsequently, the tumor cells settle in the parenchyma of target organs(especially the lung), where they create metastatic deposits[7].

Fig. 3

Comparison of migratory response of different cells to ECM proteins. Two AdCC cell lines(ACCS and ACCT)and two SCC cell lines(NA and SCCTF)were used for the haptotaxis assay using modified Boyden chambers. Briefly, polycarbonate filters were pre-coated with 10 μg/ml of the indicated ECM on the side facing the lower wells. Cells(1 × 105) were seeded in the upper compartments and migratory cells on the bottom of the filter were counted. The relative migrations of AdCC cell lines to type I and IV collagens are 10- and 5-fold those of the SCC cell lines NA and SCCTF, respectively. Further blocking experiments with specific antibodies showed that the enhanced migration of AdCC cells to the collagens was significantly and exclusively inhibited by anti-α2 integrin antibody(not shown).

Fig. 4

uPAR interactions with uPA, integrin, and ECM in AdCC cell invasion. A, uPAR is a glycosyl phosphatidylinositol(GPI) -anchored protein consisting of three homologous domains : D1, D2, and D3. D1 binds various proteins, such as uPA, PAI-1, and vitronectin, a type of ECM. uPA bound to uPAR exhibits enhanced proteolytic activity and directly activates plasminogen and matrix metalloproteases, which results in enhanced ECM degradation. The uPA-uPAR interaction also mediates several signaling events, regulates cell adhesion on the ECM, modulates integrin activity, and triggers cell migration. AdCC cells overexpress uPAR, which co-localizes with integrin, paxillin and vinculin on focal adhesions following collagen stimulation. B, CFP-uPAR overexpression leads to increased formation of lamellipodia, in which CFP-uPAR is concentrated(white arrowhead). C, Immunoprecipitation study demonstrates the effect of uPAR down-regulation on the association of focal adhesion components. ACCS-AS cells, transfected with the vector expressing uPAR antisense RNA, and control cloned ACCS-zeo cells, transfected with empty vector that expressed an amount of uPAR protein and mRNA comparable to that of parental ACCS, were plated on fibronectin(FN)or collagen I(COLL I). The cell protein complexes were immunoprecipitated using anti-paxillin antibody and then examined by Western blotting with antibody to uPAR. Novel interaction between paxillin and uPAR is found in AdCC-zeo cells induced by type I collagen stimulation. D, Immunocytochemical staining shows the colocalization of uPAR with vinculin on type IV collagen.

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Oral Science International Vol. 2, No. 2

inhibits invasion and metastasis is poorly understood.

tempted to model the events that occur after tumor cells

Our immunoprecipitation experiments showed novel in-

intravasate into the bloodstream. We showed that the

teractions between α2β1 integrin or paxillin and uPAR

adhesion of Acc-M cells to human umbilical vein endo-

in AdCC cells induced by type I collagen stimulation.

thelial cells is greater than that of non-metastatic AdCC

Therefore, in order to elucidate the association of uPAR

cells, and that the interaction between tumor cells and

with collagen-induced events, we established an ACCS-

vascular endothelial cells is mediated through adhesion

AS cell line transfected with a vector expressing anti-

molecules, including E- and P-selectins69. These are ex-

sense uPAR RNA.

uPAR expression in these cells is

pressed on stimulated endothelial cells and activated

about 10% that of parental ACCS cells at both the pro-

platelets and react with selectin glycoprotein ligand,

tein and mRNA levels. Collagen-stimulated migration

which mediates leukocyte rolling on stimulated endo-

and focal adhesion assembly of α2 integrin, vinculin,

thelial cells and the heterotypic aggregation of activated

and paxillin were strongly reduced in ACCS-AS cells.

platelets on leukocytes. A recent study suggested that

Furthermore, down-regulation of uPAR led to conforma-

P-selectin binds to cancer cells, including Acc-M, in vitro

tional changes in focal adhesions and actin organization

and promotes the growth and metastasis of human car-

(reduced stress fibers). The expression and distribu-

cinomas in vivo and sulfation was also shown to be es-

tion of α2 integrin were obscured in ACCS-AS cells plat-

sential for Acc-M cell adhesion to P-selectin70.

ed on any ECM, and there were fewer focal adhesions,

 Although the molecular mechanisms of AdCC cells

as shown by the staining pattern for paxillin. This sug-

are complex, it seems likely that metastasis is regulated

gests that uPAR plays a role in regulating the recruit-

by interactions of adhesion molecules and proteases.

ment of α2 integrin to focal adhesions, and in their as-

Further studies are required to resolve the complex

sembly, and that migration of AdCC cells to types I and

mechanisms.

IV collagens is due to the overexpression of uPAR. Acknowledgment

5)Acc-M is a model cell line for examining the

 I would like to thank Dr Seki(especially) , Dr Sugiura, Dr

mechanism of lung metastasis

Ishibashi and Dr Kurahara in our laboratory for their kind as-

 In contrast to oral SCCs, which metastasize lymphati-

sistance in preparing figure illustrations and photographs.

cally, AdCCs undergo blood-borne metastasis and in most cases metastasize to the lungs. Although the underlying molecular mechanisms are complex, it is clear that metastasis requires a cascade of sequential steps involving the coordination of adhesion, motility, and

Reference 1. Tomich C.E. : Adenoid cystic carcinoma. In : G.L.Ellis, P.L.Auclair, D.R.Gnepp(Eds.)Surgical pathology of the salivary glands. WB Saunders Company, Philadelphia 333-349, 1991.

growth66. After tumor cells intravasate into the blood-

2. Brakebusch C., Bouvard D., Stanchi F., Sakai T., and

stream, they evade innate immune surveillance, adhere

Fassler R. : Integrins in invasive growth. J Clin Invest

to vascular endothelial cells of distant organs, and ex-

109:999-1006, 2002.

travasate into those tissues. Subsequently, the malig-

3. Seiki M. : Membrane-type 1 matrix metalloproteinase : a

nant cells settle in the parenchyma of target organs

key enzyme for tumor invasion. Cancer Lett 194:1-11,

67

where they create metastatic deposits .

2003.

 The well-characterized lung metastatic AdCC cell line

4. Chapman H.A., and Wei Y. : Protease crosstalk with integ-

Acc-M was established by repeated isolation from lung

rins : the urokinase receptor paradigm. Thromb Haemost

metastatic foci of Acc-2 cells38. Cloning of the cells re-

86:124-129, 2001.

vealed a close relationship between metastasis and platelet aggregation activity(Fig. 2). Acc-M-GFP expresses green fluorescence protein(GFP)and is useful

5. Sidenius N., and Blasi F. : The urokinase plasminogen activator system in cancer : recent advances and implication for prognosis and therapy. Cancer Metastasis Rev 22: 205-222, 2003.

for the visualization of metastatic foci in vivo. Injection

6. Ossowski L., and Aguirre-Ghiso J.A. : Urokinase receptor

of Acc-M-GFP cells into the tail vein of athymic nude

and integrin partnership : coordination of signaling for

mice resulted in metastasis to multiple sites, including

cell adhesion, migration and growth. Curr Opin Cell Biol

lung, muscle, bladder, and bone68. This experiment at-

12:613-620, 2000.

November, 2005

Adenoid Cystic Carcinoma

7. Preissner K.T., Kanse S.M., and May A.E. : Urokinase receptor : a molecular organizer in cellular communication. Curr Opin Cell Biol 12:621-628, 2000.

77

patterns. Cancer 57:519-524, 1986. 23. Garden A.S., Weber R.S., Morrison W.H., Ang K.K., and Peters L.J. : The influence of positive margins and nerve

8. Andreasen P.A., Kjoller L., Christensen L., and Duffy M.J. :

invasion in adenoid cystic carcinoma of the head and neck

The urokinase-type plasminogen activator system in can-

treated with surgery and radiation. Int J Radiat Oncol

cer metastasis : a review. Int J Cancer 72:1-22, 1997.

Biol Phys 32:619-626, 1995.

9. Shirasuna K., Saka M., Hayashido Y., Yoshioka H., Sugiu-

24. Prokopakis E.P., Snyderman C.H., Hanna E.Y., Carrau

ra T., and Matsuya T. : Extracellular matrix production

R.L., Johnson J.T., and D'Amico F. : Risk factors for local

and degradation by adenoid cystic carcinoma cells : partici-

recurrence of adenoid cystic carcinoma : the role of postop-

pation of plasminogen activator and its inhibitor in matrix

erative radiation therapy. Am J Otolaryngol 20:281-

degradation. Cancer Res 53:147-152, 1993. 10. Blasi F., and Carmeliet P. : uPAR : a versatile signalling orchestrator. Nat Rev Mol Cell Biol 3:932-943, 2002. 11. Hynes R.O. : Integrins : versatility, modulation, and signaling in cell adhesion. Cell 69:11-25, 1992.

286, 1999. 25. Prott F.J., Micke O., Haverkamp U., Willich N., Schuller P., and Potter R. : Results of fast neutron therapy of adenoid cystic carcinoma of the salivary glands. Anticancer Res 20:3743-3749, 2000.

12. Dedhar S., and Hannigan G.E. : Integrin cytoplasmic in-

26. Brackrock S., Krull A., Roser K., Schwarz R., Riethdorf L.,

teractions and bidirectional transmembrane signalling.

and Alberti W. : Neutron therapy, prognostic factors and

Curr Opin Cell Biol 8:657-669, 1996.

dedifferentiation of adenoid cystic carcinomas(ACC)of

13. Miyamoto S., Akiyama S.K., and Yamada K.M. : Synergis-

salivary glands. Anticancer Res 25:1321-1326, 2005.

tic roles for receptor occupancy and aggregation in integ-

27. Chen J.C., Gnepp D.R., and Bedrossian C.W. : Adenoid

rin transmembrane function. Science 267:883-885,

cystic carcinoma of the salivary glands : an immunohisto-

1995.

chemical analysis. Oral Surg Oral Med Oral Pathol 65:

14. Wehrle-Haller B., and Imhof B.A. : Actin, microtubules and focal adhesion dynamics during cell migration. Int J Biochem Cell Biol 35:39-50, 2003. 15. Petit V., and Thiery J.P. : Focal adhesions : structure and dynamics. Biol Cell 92:477-494, 2000. 16. Turner C.E. : Paxillin interactions. J Cell Sci 113 Pt 23: 4139-4140, 2000.

316-326, 1988. 28. Hoshino M., and Yamamoto I. : Ultrastructure of adenoid cystic carcinoma. Cancer 25:186-198, 1970. 29. Perzin K.H., Gullane P., and Clairmont A.C. : Adenoid cystic carcinomas arising in salivary glands : a correlation of histologic features and clinical course. Cancer 42:265282, 1978.

17. Brown M.C., Perrotta J.A., and Turner C.E. : Serine and

30. Caselitz J., Becker J., Seifert G., Weber K., and Osborn M. :

threonine phosphorylation of the paxillin LIM domains

Coexpression of keratin and vimentin filaments in adenoid

regulates paxillin focal adhesion localization and cell ad-

cystic carcinomas of salivary glands. Virchows Arch A

hesion to fibronectin. Mol Biol Cell 9:1803-1816, 1998.

Pathol Anat Histopathol 403:337-344, 1984.

18. Greiner T.C., Robinson R.A., and Maves M.D. : Adenoid

31. Caselitz J., Osborn M., Hamper K., Wustrow J., Rauchfuss

cystic carcinoma. A clinicopathologic study with flow cyto-

A., and Weber K. : Pleomorphic adenomas, adenoid cystic

metric analysis. Am J Clin Pathol 92:711-720, 1989.

carcinomas and adenolymphomas of salivary glands anal-

19. Spiro R.H., Huvos A.G., and Strong E.W. : Adenoid cystic

ysed by a monoclonal antibody against myoepithelial/basal

carcinoma of salivary origin. A clinicopathologic study of

cells. An immunohistochemical study. Virchows Arch A

242 cases. Am J Surg 128:512-520, 1974.

Pathol Anat Histopathol 409:805-816, 1986.

20. Sung M.W., Kim K.H., Kim J.W., Min Y.G., Seong W.J.,

32. Shirasuna K., Watatani K., Furusawa H., Saka M., Morio-

Roh J.L., Lee S.J., Kwon T.K., and Park S.W. : Clinico-

ka S., Yoshioka H., and Matsuya T. : Biological character-

pathologic predictors and impact of distant metastasis

ization of pseudocyst-forming cell lines from human ade-

from adenoid cystic carcinoma of the head and neck. Arch

noid cystic carcinomas of minor salivary gland origin.

Otolaryngol Head Neck Surg 129:1193-1197, 2003.

Cancer Res 50:4139-4145, 1990.

21. Khan A.J., DiGiovanna M.P., Ross D.A., Sasaki C.T., Cart-

33. Spiro R.H., and Huvos A.G. : Stage means more than

er D., Son Y.H., and Haffty B.G. : Adenoid cystic carcino-

grade in adenoid cystic carcinoma. Am J Surg 164:623-

ma : a retrospective clinical review. Int J Cancer 96:

628, 1992.

149-158, 2001. 22. Matsuba H.M., Spector G.J., Thawley S.E., Simpson J.R.,

34. Cheng J., Saku T., Okabe H., and Furthmayr H. : Basement membranes in adenoid cystic carcinoma. An immu-

Mauney M., and Pikul F.J. : Adenoid cystic salivary gland

nohistochemical study. Cancer 69:2631-2640, 1992.

carcinoma. A histopathologic review of treatment failure

35. Shirasuna K. Kawamoto M., Saka M., Watatani K., Furu-

78

Oral Science International Vol. 2, No. 2

sawa H., Urade M., and Matsuya T. : Growth pattern of

47. Stetler-Stevenson W.G., Aznavoorian S., and Liotta L.A. :

adenoid cystic carcinomas. Jpn J Oral Maxillofac Surg

Tumor cell interactions with the extracellular matrix dur-

36:2538-2543, 1990.

ing invasion and metastasis. Annu Rev Cell Biol 9:541-

36. Shirasuna K., Furusawa H., Morioka S., Watatani K., and

573, 1993.

Matsuya T. : Different contents of glycosaminoglycans in a

48. Baramova E.N., Bajou K., Remacle A., L'Hoir C., Krell

human neoplastic salivary duct cell line and its subclone

H.W., Weidle U.H., Noel A., and Foidart J.M. : Involve-

with a myoepithelial phenotype. Virchows Arch B Cell

ment of PA/plasmin system in the processing of pro-

Pathol Incl Mol Pathol 57:175-180, 1989.

MMP-9 and in the second step of pro-MMP-2 activation.

37. He R.G. Z.X., Zhou X.J., Wang Z., Zhang X.L., Qiu W.L., et

FEBS Lett 405:157-162, 1997.

al. : The establishment of cell lines of adenoid cystic carci-

49. Franca C.M., Jaeger R.G., Freitas V.M., Araujo N.S. and

noma of human salivary glands(Acc-2, Acc-3)and a

Jaeger M.M. : Effect of N-CAM on in vitro invasion of hu-

study of morphology. West Chin J Stomatol 6:1-4,

man adenoid cystic carcinoma cells. Oral Oncol 37:638-

1988.

642, 2001.

38. Guan X.F., Qiu W.L., He R.G., and Zhou X.J. : Selection of

50. Shirasuna K., Sakai T., Sugiura T., and Matsuya T. : Char-

adenoid cystic carcinoma cell clone highly metastatic to

acterization of adenoid cystic carcinoma cells : Invasion

the lung : an experimental study. Int J Oral Maxillofac

mechanism. Head and Neck Cancer 22:7-12, 1996.

Surg 26:116-119, 1997.

51. Li C.-Y., Abu-Ali S., Sugiura T., Shiratsuchi T., Sasaki M.,

39. Abu-Ali S., Sugiura T., Takahashi M., Shiratsuchi T., Ikari

and Shirasuna K. : Integrin expression and migration of

T., Seki K., Hiraki A., Matsuki R., and Shirasuna K. : Ex-

adenoid cystic carcinoma cells in response to basement

pression of the urokinase receptor regulates focal adhesion

membrane components. Oral Sci Int 1:22-29, 2004.

assembly and cell migration in adenoid cystic carcinoma

52. Piguet P.F., Vesin C., Donati Y., Tacchini-Cottier F., Belin

cells. J Cell Physiol 203:410-419, 2004.

D., and Barazzone C. : Urokinase receptor(uPAR, CD87)

40. Cheng J., Irie T., Munakata R., Kimura S., Nakamura H.,

is a platelet receptor important for kinetics and TNF-in-

He R.G., Lui A.R., and Saku T. : Biosynthesis of basement

duced endothelial adhesion in mice. Circulation 99:

membrane molecules by salivary adenoid cystic carcinoma

3315-3321, 1999.

cells : an immunofluorescence and confocal microscopic study. Virchows Arch 426:577-586, 1995.

53. Memarzadeh S., Kozak K.R., Chang L., Natarajan S., Shintaku P., Reddy S.T., and Farias-Eisner R. : Urokinase

41. Laiho M., and Keski-Oja J. : Growth factors in the regula-

plasminogen activator receptor : Prognostic biomarker for

tion of pericellular proteolysis : a review. Cancer Res 49:

endometrial cancer. Proc Natl Acad Sci USA 99:10647-

2533-2553, 1989.

10652, 2002.

42. Rosenthal E.L., Johnson T.M., Allen E.D., Apel I.J., Pun-

54. Doerr T.D., Marentette L.J., Flint A., and Elner V. : Uroki-

turieri A., and Weiss S.J. : Role of the plasminogen activa-

nase-type plasminogen activator receptor expression in

tor and matrix metalloproteinase systems in epidermal

adenoid cystic carcinoma of the skull base. Arch Otolaryn-

growth factor- and scatter factor-stimulated invasion of

gol Head Neck Surg 129:215-218, 2003.

carcinoma cells. Cancer Res 58:5221-5230, 1998.

55. Hedberg K.K., Stauff C., Hoyer-Hansen G., Ronne E., and

43. Hayashido Y., Shirasuna K., Sugiura T., Nakashima M.,

Griffith O.H. : High-molecular-weight serum protein com-

and Matsuya T. : Effect of dexamethasone on invasion of

plexes differentially promote cell migration and the focal

human squamous cell carcinoma cells into collagen gel.

adhesion localization of the urokinase receptor in human

Cancer Lett 108:81-86, 1996.

glioma cells. Exp Cell Res 257:67-81, 2000.

44. Shiratsuchi T., Ishibashi H., and Shirasuna K. : Inhibition

56. Wei Y., Eble J.A., Wang Z., Kreidberg J.A., and Chapman

of epidermal growth factor-induced invasion by dexameth-

H.A. : Urokinase receptors promote beta1 integrin func-

asone and AP-1 decoy in human squamous cell carcinoma

tion through interactions with integrin alpha3beta1. Mol

cell lines. J Cell Physiol 193:340-348, 2002.

Biol Cell 12:2975-2986, 2001.

45. Sato H., and Seiki M. : Regulatory mechanism of 92 kDa

57. Wickstrom S.A., Veikkola T., Rehn M., Pihlajaniemi T., Al-

type IV collagenase gene expression which is associated

italo K., and Keski-Oja J. : Endostatin-induced modulation

with invasiveness of tumor cells. Oncogene 8:395-405,

of plasminogen activation with concomitant loss of focal

1993.

adhesions and actin stress fibers in cultured human endo-

46. Sato H., Takino T., Okada Y., Cao J., Shinagawa A., Yama-

thelial cells. Cancer Res 61:6511-6516, 2001.

moto E., and Seiki M. : A matrix metalloproteinase ex-

58. Shirasuna K. : Search for molecular targets involved in

pressed on the surface of invasive tumour cells. Nature

oral tumor invasion. Head and Neck Cancer 29:487-

370:61-65, 1994.

492, 2003.

November, 2005

Adenoid Cystic Carcinoma

59. Ghosh S., Brown R., Jones J.C., Ellerbroek S.M., and

79

rangements and increased cell motility induced by uroki-

Stack M.S. : Urinary-type plasminogen activator(uPA)

nase-type plasminogen activator receptor binding to vitro-

expression and uPA receptor localization are regulated by

nectin. J Cell Biol 152:1145-1157, 2001.

alpha 3beta 1 integrin in oral keratinocytes. J Biol Chem 275:23869-23876, 2000. 60. van der Pluijm G., Sijmons B., Vloedgraven H., van der Bent C., Drijfhout J.W., Verheijen J., Quax P., Karperien

65. Wang Y., Liang X., Wu S., Murrell G.A., and Doe W.F. : Inhibition of colon cancer metastasis by a 3'- end antisense urokinase receptor mRNA in a nude mouse model. Int J Cancer 92:257-262, 2001.

M., Papapoulos S., and Lowik C. : Urokinase-receptor/inte-

66. Liotta L.A., Steeg P.S., and Stetler-Stevenson W.G. : Can-

grin complexes are functionally involved in adhesion and

cer metastasis and angiogenesis : an imbalance of positive

progression of human breast cancer in vivo. Am J Pathol 159:971-982, 2001.

and negative regulation. Cell 64:327-336, 1991. 67. Koike C., Watanabe M., Oku N., Tsukada H., Irimura T.,

61. Yu W., Kim J., and Ossowski L. : Reduction in surface uro-

and Okada S. : Tumor cells with organ-specific metastatic

kinase receptor forces malignant cells into a protracted

ability show distinctive trafficking in vivo : analyses by

state of dormancy. J Cell Biol 137:767-777, 1997.

positron emission tomography and bioimaging. Cancer

62. Mohan P.M., Chintala S.K., Mohanam S., Gladson C.L., Kim E.S., Gokaslan Z.L., Lakka S.S., Roth J.A., Fang B.,

Res 57:3612-3619, 1997. 68. Xiong T., Zhang Z., Liu B.F., Zeng S., Chen Y., Chu J., and

Sawaya R., Kyritsis A.P., and Rao J.S. : Adenovirus-medi-

Luo Q. : In vivo optical imaging of human adenoid cystic

ated delivery of antisense gene to urokinase-type plasmin-

carcinoma cell metastasis. Oral Oncol 41:709-715, 2005.

ogen activator receptor suppresses glioma invasion and

69. Seki K., Ishii K., Sugiura T., Takahashi M., Inoue Y., and

tumor growth. Cancer Res 59:3369-3373, 1999. 63. Chintala S.K., Mohanam S., Go Y., Venkaiah B., Sawaya R., Gokaslan Z.L., and Rao J.S. : Altered in vitro spreading

Shirasuna K. : An adenoid cystic carcinoma cell possessing high metastatic activity in response to TNF-α. Oral Sci Int  2:36-44, 2005.

and cytoskeletal organization in human glioma cells by

70. Ma Y.Q., and Geng J.G. : Obligatory requirement of sul-

down-regulation of urokinase receptor. Mol Carcinog

fation for P-selectin binding to human salivary gland car-

20:355-365, 1997.

cinoma Acc-M cells and breast carcinoma ZR-75-30 cells. J

64. Kjoller L., and Hall A. : Rac mediates cytoskeletal rear-

Immunol 168:1690-1696, 2002.