Oral Science International, November 2005, p.69-79 Copyright © 2005, Japanese Stomatology Society. All Rights Reserved.
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,
Oral Science International Vol. 2, No. 2
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,
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
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 .
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
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-
Adenoid Cystic Carcinoma
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
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
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
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
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-
controlled . The most frequent site of metastasis is the
al transition from basal to luminal cells, equivalent to
Oral Science International Vol. 2, No. 2
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） .
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
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-
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
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-
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
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.
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.
Adenoid Cystic Carcinoma
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-
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
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
duction of PAI-1 expression . Rosenthal et al.
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
. 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
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-
nerve and endothelial sheaths
, the frequency of re-
ant of green ﬂuorescent protein（CFP）.
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-
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
Oral Science International Vol. 2, No. 2
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
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,
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） .
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］.
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）.
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.
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
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
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