* Pathology Division, National Cancer Center Research Institute, Tokyo 104, Japan; Department of Oral and Maxillofacial
Surgery, Tokyo Medical College, Tokyo 160, Japan; and § Hirohashi Cell Configuration Project, ERATO, Japan Scientific and
Technology Corporation (JST), Tsukuba 300-26, Japan
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Abstract |
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Regulation of the actin cytoskeleton may
play a crucial role in cell motility and cancer invasion.
We have produced a monoclonal antibody (NCC-
Lu-632, IgM, k) reactive with an antigenic protein that
is upregulated upon enhanced cell movement. The cDNA
for the antigen molecule was found to encode a novel
isoform of nonmuscle -actinin. This isoform (designated actinin-4) was concentrated in the cytoplasm
where cells were sharply extended and in cells migrating and located at the edge of cell clusters, but was absent from focal adhesion plaques or adherens junctions,
where the classic isoform (actinin-1) was concentrated.
Actinin-4 shifted steadily from the cytoplasm to the nucleus upon inhibition of phosphatidylinositol 3 kinase
or actin depolymerization. The cytoplasmic localization of actinin-4 was closely associated with an infiltrative
histological phenotype and correlated significantly with
a poorer prognosis in 61 cases of breast cancer. These
findings suggest that cytoplasmic actinin-4 regulates the
actin cytoskeleton and increases cellular motility and
that its inactivation by transfer to the nucleus abolishes
the metastatic potential of human cancers.
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Introduction |
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EVEN after the successful removal of a primary cancer
by surgery, recurrences often develop at distant
sites. Cancer metastasis is a complicated process
involving local invasion by cancer cells, infiltration into
vascular and lymphatic vessels, survival in the circulation,
extravasation, and growth at secondary sites. In this sequence of events, local invasion may play an important role as the initial step in cancer metastasis (Filder et al., 1978; Nicolson, 1988
; Zetter, 1990
; Aznavoorian et al.,
1993
). The mechanisms by which cancer invasion occurs
are poorly understood, but one of the requirements is enhancement of cell motility. In fact, enhanced movement of
cancer cells has been reported to be correlated with metastatic potential in animal models and with a poor prognosis in human cancers (Hosaka et al., 1978
; Haemmerli and
Strauli, 1981
; Partin et al., 1989
).
Although cell motility is regulated by several cellular
and extracellular factors, the bundling and subcellular distribution of the actin cytoskeleton directly determines cell
motility (Stossel, 1993; Lauffenburger and Horwitz, 1996
;
Cramer et al., 1997
).
-Actinin is thought to cross-link actin filaments and connect the actin cytoskeleton to the cell
membrane. So far, three isoforms of human
-actinin have
been identified: nonmuscle actinin-1, muscle actinin-2, and
muscle actinin-3 (Millake et al., 1989
; Youssoufian et al., 1990
; Beggs et al., 1992
). Nonmuscle actinin-1 has been reported to associate with cell adhesion molecules, such as
integrin
1 and
-catenin, and is believed to play an important role in stabilizing cell adhesion and regulating cell
shape and cell motility (Otey et al., 1990
, 1993
; Glück et al.,
1993
; Glück and Ben-Ze'ev, 1994
; Knudsen et al., 1995
).
In this study, we looked for a new molecular marker that is associated with cell motility and cancer invasion and obtained an mAb. cDNA for the antigen was found to encode a novel isoform of nonmuscle actinin. Notably, this actinin isoform was found to be translocated into the nucleus in a certain population of human cancer cells and in several cell lines. We report here that the subcellular localization of this newly identified actinin may indicate the enhanced motility of cancer cells and predict the metastatic potential of human cancer.
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Materials and Methods |
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Cell Culture and Chemicals
The human giant cell-lung cancer cell line, Lu-65, was established previously in our laboratory (Yamada et al., 1985). Primary cultured human
foreskin keratinocytes (HFK)1 were purchased from Seikagaku Co. (Tokyo, Japan) and were cultured as instructed by the supplier. Primary normal human uterine endometrial fibroblasts were isolated and cultured as
described previously (Zhang et al., 1995
). PC-10, a human squamous lung
cancer cell line, was provided by Dr. Y. Hayata (Tokyo Medical College,
Tokyo, Japan). A-431 is a human vulvar epidermoid cancer cell line (Giard et al., 1973
), and KU7 is a urinary bladder cancer cell line. TE 4, TE 6, TE 7, and TE 10 were derived from esophageal cancers (Nishihira et al.,
1993
), and MCF7 and R27 were derived from breast cancers (Westley,
1979
; Cavailles et al., 1988
). The oral floor cancer cell line IMC2, normal
embryonic lung fibroblast MRC5, and colon cancer cell line WiDr were
obtained from Riken Cell Bank (Tsukuba, Japan). Colon cancer cell line
SW480 was obtained from the American Type Culture Collection (Rockville, MD).
HFK were treated with 50 ng/ml wortmannin (Sigma Chemical Co., St. Louis, MO) for 15 min and then washed. After incubation for 24 h, the cells were examined by immunofluorescence microscopy as described below. For actin depolymerization, the cells were treated with 2 µg/ml cytochalasin D (Sigma Chemical Co.) for 2 h.
Production of Antibodies
BALB/c mice were immunized with a lysate of Lu-65 cells, and hybridomas were produced as described previously (Hirohashi et al., 1984). The
hybridoma supernatants were then screened immunohistochemically for
reactivity with acetone-fixed paraffin-embedded human tissues, as described below.
Rabbit polyclonal anti-actinin-4 antibody was raised against a KLH-conjugated synthetic peptide, MGDYMAQEDDWC (containing amino acids 1-11, Fig. 1), and affinity-purified.
|
cDNA Cloning and DNA Sequencing
A lambda gt11 cDNA expression library of primary cultured HFK
(CLONTECH Laboratories, Inc., Palo Alto, CA) was immunoscreened with mAb NCC-Lu-632, as described previously (Young and Davis, 1983),
and a positive 950-bp cDNA clone (ck-20-2) was isolated. For isolation of
full-length cDNA of actinin-4 and -1, a cDNA library of mesothelioma
MS-1/CDDP cells was screened with synthetic oligonucleotides specific to
each actinin. A 3.5-kb actinin-4 cDNA clone, near full-length as determined by Northern blot analysis (Fig. 2 B), was isolated, and both strands
were sequenced using an autosequencer (model ABI 377; Perkin Elmer,
Foster City, CA).
|
Northern Blot Analysis
Multiple tissue Northern blots I and II were purchased from CLONTECH
Laboratories and hybridization was performed using a 32P-labeled oligonucleotide probe (5'-CGCTTCTGAGTTAGGGCCCCCA-3') specific to
actinin-4, as described previously (Sambrook et al., 1989). The quality and
quantity of electrophoresed mRNA was determined by rehybridization of
the same blot with a human
-actin cDNA probe.
Extraction of Cell Lysate and Western Blot Analysis
Cells were extracted with lysis buffer (10 mM Hepes, pH 7.4, 150 mM
NaCl, 1 mM EDTA, 1% Triton X-100, 1% NP-40, 1 mg/ml NaN3, 0.5 mM
Na3Vo4, 10 nM -glycerophosphate, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml
pepstatin A) on ice for 30 min before centrifugation (12,000 g, 15 min).
Cell lysates (50 µg protein) were separated by SDS-PAGE (Laemmli,
1970
) and transferred to Hybond-polyvinyl difluoride membranes (Amersham International, Buckinghamshire, UK) (Towbin et al., 1979
). After
incubation with antibodies at 4°C overnight, the blots were detected using
a horseradish peroxidase-conjugated anti-mouse IgG+IgM (IBL, Fujioka, Japan) or anti-rabbit Ig antibody and ECL Western blotting detection reagents (Amersham International), as instructed by the suppliers.
For blotting of whole cell lysates, cells were lysed directly with Laemmli
buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 5% glycerol, 715 mM 2-mercaptoethanol, 0.0025% bromophenol blue) (Harlow and Lane, 1988
).
Glutathione S-Transferase Fusion Protein Production and In Vitro Translation
A cDNA fragment of actinin-4 (encoding amino acids 410-664) and the
corresponding site of actinin-1 (amino acids 418-672) were amplified by
PCR and subcloned into pGEX plasmids (Pharmacia Biotech, Uppsala,
Sweden). Glutathione S-transferase (GST) fusion proteins expressed in
Escherichia coli were affinity-purified on glutathione-Sepharose 4B
(Pharmacia Biotech) as described previously (Shibata et al., 1996).
L-[35S]methionine (Amersham International) labeled polypeptides
were synthesized from cDNA clones containing the entire coding regions
for actinin-1 and -4, using the TNT reticulocyte lysate system (Promega
Corp., Madison, WI) as instructed by the supplier. The synthesized
polypeptides were analyzed by SDS-PAGE and autoradiography as described previously (Yamada et al., 1995).
Actin-binding Assay
Purified chicken gizzard actin (Sigma Chemical Co.) was coupled to
CNBr-activated Sepharose-4B beads (Pharmacia Biotech) (Prendergast and Ziff, 1991). The beads were incubated with 35S-incorporated in vitro
translation products in lysis buffer overnight at 4°C. After extensive washing with lysis buffer, the beads were boiled in Laemmli buffer and analyzed by SDS-PAGE and autoradiography (Yamada et al., 1995
).
A cell lysate of the lung cancer cell line PC-10 was also incubated overnight at 4°C with the actin-coupled beads. After thorough washing, the beads were boiled in Laemmli buffer and analyzed by immunoblotting as described above.
Immunoprecipitation
In vitro translation products of actinins were incubated with a monoclonal
antibody against chicken -actinin (BM-75.2, IgM, k; Sigma Chemical
Co.), anti-actinin-4 polyclonal antibody, normal mouse IgM, or normal
rabbit IgG (Sigma Chemical Co.) overnight at 4°C and precipitated with
anti-mouse IgM agarose (for mouse antibodies) (Sigma Chemical Co.) or
protein G plus agarose (for rabbit antibodies) (Santa Cruz Biotechnology,
Santa Cruz, CA). SDS-PAGE and autoradiography were carried out as
described previously (Yamada et al., 1995
).
Immunofluorescence Microscopy and "Wound" Assay
Cells were cultured on glass coverslips, fixed with 4% paraformaldehyde and 2% sucrose in PBS, and made permeable with 0.2% Triton X-100. After incubation with antibodies, actinins were detected with biotinylated anti-mouse IgM or biotinylated anti-rabbit IgG and Texas red-avidin D or FITC-conjugated avidin D (Vector Laboratories, Burlingame, CA). Actin filaments were visualized with FITC-phalloidin (Sigma Chemical Co.), and the cells were examined with a Zeiss LSM410 microscope (Thornwood, NY).
Cells were grown to confluency on glass coverslips, and a "wound" was
then introduced in the monolayers with a plastic pipette tip (Glück et al.,
1993; Glück and Ben-Ze'ev, 1994
). After incubation for 24 h, the cells
were fixed and examined as described above.
Immunohistochemistry
Acetone-fixed paraffin-embedded human tissues (Sato et al., 1986) were
selected from the surgical pathology file of the National Cancer Center
Hospital (Tokyo, Japan). 5-µm thin sections were stained with mAb
NCC-Lu-632 using an immunoperoxidase avidin-biotin complex (ABC)
kit (Vector Laboratories), as described previously (Sato et al., 1986
). Histological subtyping of breast cancer was done by two independent certified pathologists, according to the criteria described previously (Rosen
and Oberman, 1992
).
Statistical Analysis
The postsurgical survival curves for a total of 61 patients with stages I and
II breast cancer (Rosen and Oberman, 1992) were expressed as described
by Kaplan and Meier (1958)
. Statistical significance was determined by
the generalized Wilcoxon test.
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Results |
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Molecular Cloning of Actinin-4 cDNA
mAb NCC-Lu-632 (IgM, k) was selected on the basis of its intriguing immunohistochemical reactivity, as described below. A cDNA clone encoding the antigen molecule recognized by the mAb was isolated by immunoscreening of the HFK lambda gt11 cDNA library.
The 3.5-kb cDNA clone contained a 2,652-bp open
reading frame encoding 884 amino acids (Fig. 1), with a
predicted molecular mass of 102 kD. The mAb NCC-Lu-632 reacted consistently with the ~100-kD antigen
molecule in immunoblot analyses (Fig. 2 A), and in vitro
translation of the full-length cDNA yielded a major protein of ~100 kD (Fig. 3 C). This coding region and the deduced
amino acid sequence showed a high degree of similarity to
human -actinin-1 (Millake et al., 1989
; Youssoufian et al.,
1990
) (80.0% nucleotide and 86.7% amino acid similarity).
It contained structures conserved among
-actinin family
members, including the actin-binding domain (Kuhlman et
al., 1992
) (amino acids 111-125; Fig. 1), pleckstrin-homology (PH) domain containing a phosphatidylinositol 4, 5-biphosphate (PIP2)-binding site (Fukami et al., 1996
) (amino acids
150-165), and two EF-hand calcium regulation domains
(Witke et al., 1993
; Imamura et al., 1994
) (amino acids
742-770 and 783-811). Following the nomenclature proposed by Beggs et al. (1992)
, this gene was termed actinin-4.
|
Expression of Actinin-4 mRNA in Normal and Neoplastic Cells
Northern hybridization was carried out with an actinin- 4-specific 22-bp oligonucleotide probe to avoid cross- hybridization with actinin-1. As shown in Fig. 2 B, intense expression of 3.5-kb actinin-4 mRNA was detected in the ovary (Fig. 2 B, lane 13) and colon (lane 15), and faint expression was detected in all tissues examined. No gross structural alterations of actinin-4 mRNA were detected in any of the human carcinoma cell lines examined (data not shown).
Specificity of Antibodies
To determine the specific reactivity of mAb NCC-Lu-632
with actinin-4, amino acids 410-664 of actinin-4 and a corresponding site of actinin-1 (Millake et al., 1989) (amino
acids 418-672) were expressed as a GST fusion protein in
E. coli. Immunoblot analysis revealed that the mAb reacted only with actinin-4 GST fusion protein, and not with
actinin-1 GST fusion protein or GST alone (Fig. 3 A), confirming the specificity of mAb NCC-Lu-632 for actinin-4.
Immunoprecipitation analyses revealed that an mAb against chicken actinin, BM-75.2, was reactive with the in vitro translation product of cDNA of actinin-1, but not with that of actinin-4 (Fig. 3 D).
Immunoblot analyses revealed that the polyclonal antibody raised against the NH2-terminal amino acids of actinin-4 reacted only with a single band of the same molecular weight as NCC-Lu-632 mAb (Fig. 4 A). Immunoprecipitation analyses revealed that this polyclonal antibody was reactive with the in vitro translation product of the cDNA of actinin-4, but not with that of actinin-1 (Fig. 4 B), thus confirming its specificity.
|
Actin-binding Activity In Vitro
From the deduced amino acid sequence (Fig. 1), actinin-4
was predicted to contain an actin-binding domain (Hemmings and Critchley, 1992; Kuhlman et al., 1992). To confirm this, in vitro binding experiments were performed using actin-coupled agarose beads (Prendergast and Ziff,
1991
). 35S-labeled actinin-4 recombinant protein, produced by in vitro translation, was incubated overnight with
agarose beads coupled to actin. After extensive washing,
SDS-PAGE and autoradiography revealed labeled polypeptides whose size was consistent with the predicted size
of actinin-4 (Fig. 5 A).
|
Similarly, a cell lysate of the lung cancer cell line PC-10 was incubated overnight with actin-agarose beads and washed thoroughly. Immunoblot analysis with mAb NCC-Lu-632 revealed the retention of actinin-4 on actin-agarose beads (Fig. 5 B), confirming the actin-binding activity of actinin-4.
Distinct Subcellular Localization of Two Nonmuscle Actinins
Confocal immunofluorescence microscopy revealed that
actinin-1 was localized specifically at the ends of actin
stress fibers (Fig. 6, A, C, and E) and adherens junctions
(Fig. 7 A), as described previously (Lazarides, 1975; Wehland et al., 1979; Knudsen et al., 1995
). In contrast to this
cell membrane-associated localization of actinin-1, actinin-4 protein was colocalized with actin stress fibers and
was dispersed in the cytoplasm and nucleus (Fig. 6, B, D,
and F). The specific immunocytochemical reactivity of
monoclonal antibody NCC-Lu-632 was further confirmed
by the polyclonal antibody raised against the NH2-terminal amino acids of actinin-4 (Fig. 4 C). In most cancer cell
lines including PC10, A431, SW480, TE4, TE6, TE7,
TE10, and R27, actin stress fibers were poorly developed and actinin-4 was diffusely dispersed in the cytoplasm.
Characteristically, actinin-4 was highly concentrated in the
cytoplasm where the cells were sharply extended (Fig. 8,
B, E, and H). Actinin-4 was stained intensely at the edges
of cell clusters (Fig. 8, C, F, and I). The difference in subcellular localization between these two nonmuscle isoforms suggested that they were associated with different
functions.
|
|
|
Upregulation of Actinin-4 upon Enhanced Cell Movement
From the above immunofluorescence observations, we speculated that the expression of actinin-4 was regulated dynamically by cell movement. An artificial linear defect was introduced in the confluent monolayers of A-431 and R27 cells, and we examined the cells forced to be motile by immunofluorescence microscopy. As shown in Fig. 9, actinin-4 was markedly induced in cells along the edges of the wound (Fig. 9 A) and in cells migrating into the wound (Fig. 9, B-D).
|
Nuclear Translocation of Actinin-4
In a limited number of cell lines, including the breast cancer cell line MCF7, oral floor cancer IMC2, and bladder cancer KU7, it was noticed that actinin-4 was localized exclusively in the nucleus (Fig. 7 B), in contrast to the association of actinin-1 with the cell membrane in these cells (Fig. 7 A). The nuclear staining was not due to cross-reactivity of the mAb with other nuclear proteins because NCC-Lu-632 mAb reacted only with a protein of ~100 kD in whole-cell lysates of these cell lines (Fig. 7 C).
Actinin-4 possesses a conserved PIP2-binding site. PIP2
binds -actinin through the pleckstrin homology domain
and regulates its actin-binding activity (Fukami, 1992;
Fukami et al., 1996
). PIP2 is one of the substrates of phosphatidylinositol 3 kinase (PI3 kinase). By treating HFK
with a PI3 kinase inhibitor, wortmannin (Vemuri, 1994
),
actinin-4 was steadily translocated into the nucleus (Fig.
10). A bladder cancer cell line, KU7, which is deficient in
PI3 kinase expression, also showed nuclear localization of actinin-4 without treatment (data not shown). These findings suggest that inhibition of the PI3 kinase-mediated signaling pathway is involved in the nuclear translocation of
actinin-4.
|
Cytochalasin D inhibits actin polymerization (Casella
et al., 1981). Treatment with cytochalasin D also induced
nuclear translocation (data not shown), suggesting that the
nuclear translocation of actinin-4 is also caused by loss of
its association with the cytoplasmic actin cytoskeleton.
Subcellular Distribution of Actinin-4 Predicts Poor Prognosis of Breast Cancer
The distribution of actinin-4 was determined immunohistochemically in human tissue using the mAb, NCC-Lu-632. Actinin-4 was expressed in a limited population of normal cells, including erythrocytes, endothelial cells, and epithelial cells in various tissues at their border with stromal connective tissue.
In the ducts of normal mammary glands, the epithelio-
stromal and epithelio-myoepithelial borders were stained,
but the nucleus of normal mammary gland cells did not
stain (Fig. 11 A). Scirrhous carcinoma and invasive lobular
carcinoma of the breast manifest highly infiltrative growth
into the fibrous stroma, in contrast to low-infiltrative histological types including papillotubular and solid-tubular
carcinomas (Rosen and Oberman, 1992). To determine the relationship between the growth pattern of breast cancer and the subcellular localization of actinin-4, 83 cases of
invasive breast cancer were examined immunohistochemically. The reactivity of mAb NCC-Lu-632 was classified
into three types (Table I): nuclear (type A, Fig. 11 B),
combined (type B), and cytoplasmic (type C, Fig. 10, C
and D). Histologically, all type A cases were papillotubular or solid-tubular carcinomas that showed low-infiltrative extension (20/20). In type B, the majority of cases
were papillotubular and solid tubular carcinomas (25/33)
and fewer were scirrhous carcinomas (8/33). In contrast to
these two types, most type C cases were scirrhous (15/30)
(Fig. 11 C) and invasive lobular carcinomas (6/30) (Fig. 11
D), both of which extended locally in a highly infiltrative
manner.
|
|
We then determined whether these staining patterns
were correlated with prognosis in patients with breast cancer. The relationship between the actinin-4 staining pattern and disease-free and overall survival of 61 patients
with relatively early breast cancer (clinical stages I and II
[Rosen and Oberman, 1992]) was examined. A significant difference in disease-free survival was observed between
types A+B and type C (P < 0.01) (Fig. 12 A). In addition,
a significant difference in overall survival was observed
between types A + B and type C (P < 0.01) (Fig. 12 B).
These findings suggest that the subcellular distribution of
actinin-4 may indicate the biological behavior of breast
cancer and may be considered a new risk factor for relapse.
|
![]() |
Discussion |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
-Actinin is a member of the superfamily of actin-binding
proteins that includes spectrin, filamin, dystrophin, and
fimbrin, by virtue of the similarity of their actin-binding
domains (Matsudaira, 1991
).
-Actinin binds to the integrin
1 cytoplasmic domain (Otey et al., 1990
, 1993
) and is
believed to link the actin cytoskeleton to focal adhesions
in cooperation with other cytoplasmic proteins, including
tensin, talin, and vinculin (Burridge et al., 1990
; Miyamoto
et al., 1995
). Actinin is also localized at adherens junctions
in connection with
-catenin (Knudsen et al., 1995
). Assembly of these structural proteins is believed to play an
important role in stabilizing cell adhesion (Burridge et al.,
1990
; Miyamoto et al., 1995
) and suppressing cell motility
(Glück et al., 1993
; Glück and Ben-Ze'ev, 1994
).
In this study, using a newly established mAb, we isolated a cDNA clone encoding a novel isoform of -actinin,
designated actinin-4. Immunofluorescence microscopy demonstrated that the classical isoform, actinin-1, was concentrated in actin stress fiber ends and adherens junctions. In
contrast, this novel nonmuscle
-actinin, actinin-4, binds actin filaments at a different subcellular location, thereby exerting functions distinct from those of actinin-1. Actinin-4 is highly concentrated in sharply stretched cells. In the
wound assay, the cells forced to be motile along wound
edges and cells migrating into the wound overexpressed
actinin-4. These findings suggest the involvement of actinin-4 in cell movement. Glück et al. demonstrated that the
expression of actinin-1 was reduced in SV-40-transformed
3T3 cells (Glück et al., 1993
; Glück and Ben-Ze'ev, 1994
).
Transfection with actinin-1 cDNA suppressed tumorigenicity. Transfection with actinin-1 cDNA in an antisense orientation increased cell motility, supporting the contradictory roles of the two nonmuscle isoforms with respect
to cell movement.
Various growth factors provide stimuli for cell motility
in addition to mitogenic activity (Klagsbrun, 1996). Recently, Hsu et al. (1996)
identified a new murine nonmuscle
-actinin isoform, FR-17, as a fibroblast growth factor-1-inducible gene in NIH-3T3 cells. Although it has not
been fully sequenced, the deduced COOH-terminal amino
acids (1-109; sequence data available from GenBank/
EMBL/DDBJ under accession number U41415/6) available for FR-17 showed 97.2% identity with amino acids
776-884 of human actinin-4, suggesting that this isoform
may be a murine homologue of human actinin-4. In addition to FR-17, a computer search revealed that several partial sequences identical to actinin-4 were registered in
dbEST databases. These ESTs were isolated from human
cDNAs of placenta (accession number AA368907), fetal
brain (M85377), Jurkat T-cells (AA312012), and pancreatic islets (C06273, D83843).
The level of another actin-binding protein, thymosin
15, was reported to be elevated in human metastatic
prostate cancer and correlated positively with the Gleason
score (Bao et al., 1996
). In contrast to actinins, thymosins
inhibit actin polymerization. Cell motility is regulated by
both disassembly and reformation of the actin cytoskeleton. Thymosin
15 has been considered to degrade the
static anchorage of the actin cytoskeleton and liberate actin monomers. Actinin-4 may then cross-link actin filaments and reorganize the cytoskeleton, which is essential
for cell movement. Further studies are necessary to elucidate how these molecules regulate the actin cytoskeleton
and participate in cancer invasion and metastasis.
In this study, we found that actinin-4 existed in the nucleus of a certain population of breast cancers and in several cell lines. From the deduced amino acid sequence, it
seems that actinin-4 does not possess any apparent nuclear
localization signals (Boulikas, 1993). Although the precise
mechanisms of the nuclear transition are still under investigation, we were able to reproduce the nuclear transition
of actinin-4 in tissue culture. Actinin-4 was translocated from the cytoplasm to the nucleus by treatment with the
PI3 kinase inhibitor, wortmannin (Vemuri, 1994
), or by actin depolymerization using cytochalasin D (Casella et al.,
1981
). PI3 kinase is thought to be one of the key molecules
involved in the signaling pathways of activated receptor
tyrosine kinases and regulates cell growth, motility, and
morphogenesis (Raffioni and Bradshaw, 1992
; Royal and
Park, 1995
). The nuclear localization of actinin-4 was observed mainly in low-infiltrative histological subtypes of
breast carcinoma. The abnormal activation of this enzyme
may determine the biological behavior of cancer cells and
confer metastatic potential to them.
Although histological subtyping is a common procedure in the pathological diagnosis of breast cancer, the correlation between histological subtypes and the outcome of patients in this study did not reach statistical significance (data not shown). However, we demonstrated a significantly poorer disease-free survival and overall survival in breast cancer patients with a nonnuclear localization of actinin-4 (Fig. 12). This difference was probably due to cases of papillotubular and solid-tubular carcinomas, in which the location of actinin-4 was exceptionally cytoplasmic. These cases may have had a high degree of metastatic potential that could not be detected by conventional histological examination.
From the above experimental and clinical observations, we conclude that a cytoplasmic localization may indicate active actin-bundling by actinin-4 and enhanced cell motility. Because no definite marker is currently available for assessment of the metastatic potential of cancer, the subcellular localization of the actinin-4 molecule may represent a potential new biological marker for cancer metastasis and may predict early relapse of breast cancer. Large-scale prospective analyses are necessary to examine this issue, not only for breast cancer but also for other cancers.
![]() |
Footnotes |
---|
Received for publication 21 July 1997 and in revised form 13 January 1998.
K. Honda is a recipient of a Research Resident Fellowship from the Foundation for Promotion of Cancer Research. This research was supported in part by a Grant-in-Aid for the Second Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare, Japan. ![]() |
Abbreviations used in this paper |
---|
GST, glutathione S-transferase; HFK, human foreskin keratinocyte(s); PH, pleckstrin homology; PI3, phosphatidylinositol 3; PIP2, phosphatidylinositol 4,5-biphosphate.
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Aznavoorian, S., A.N. Murphy, W.G. Stetler-Stevenson, and L.A. Liotta. 1993. Molecular aspects of tumor cell invasion and metastasis. Cancer. 71: 1368-1383 |
2. |
Bao, L.,
M. Loda,
A.P. Janmey,
R. Stewart,
B. Anand-Apte, and
B.R. Zetter.
1996.
Tymosin ![]() |
3. |
Beggs, A.H.,
T.J. Byers,
J.H.M. Knoll,
F.M. Boyce,
G.A.P. Bruns, and
L.M. Kunkel.
1992.
Cloning and characterization of two human skeletal muscle
![]() |
4. | Boulikas, T.. 1993. Nuclear localization signals (NLS). Crit. Rev. Eukaryotic Gene Expr. 3: 193-227 |
5. | Burridge, K., G. Nuckolls, C. Otey, F. Pavalko, K. Simon, and C. Turner. 1990. Actin-membrane interaction in focal adhesions. Cell Differ. Dev. 32: 337-342 |
6. | Casella, J.F., M.D. Flanagan, and S. Lin. 1981. Cytochalasin D inhibits actin polymerization and induces depolymerization of actin filaments formed during platelet shape change. Nature. 293: 302-305 |
7. | Cavailles, V., P. Augereau, M. Garcia, and H. Rochefort. 1988. Estrogens and growth factors induce the mRNA of the 52K-pro-cathepsin-D secreted by breast cancer cells. Nucleic Acids Res. 16: 1903-1919 [Abstract]. |
8. |
Cramer, L.P.,
M. Siebert, and
T.J. Mitchison.
1997.
Identification of novel
graded polarity actin filament bundles in locomoting heart fibroblasts.
J. Cell Biol.
136:
1287-1305
|
9. | Filder, I.J., D.M. Gersten, and I.R. Hart. 1978. The biology of cancer invasion and metastasis. Adv. Cancer Res. 28: 149-250 |
10. |
Fukami, K.,
K. Furuhashi,
M. Inagaki,
T. Endo,
S. Hatano, and
T. Takenawa.
1992.
Requirement of phosphatidylinositol 4,5-bisphosphate for ![]() |
11. |
Fukami, K.,
N. Sawada,
T. Endo, and
T. Takenawa.
1996.
Identification of a
phosphatidylinositol 4,5-bisphosphate-binding site in chicken skeletal muscle ![]() |
12. | Giard, D.J., S.A. Aaronson, G.J. Todaro, P. Arnstein, J.H. Kersey, H. Dosik, and W.P. Parks. 1973. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J. Natl. Cancer Inst. 51: 1417-1423 |
13. |
Glück, U., and
A. Ben-Ze'ev.
1994.
Modulation of ![]() |
14. |
Glück, U.,
D.J. Kwiatkowski, and
A. Ben-Ze'ev.
1993.
Suppression of tumorigenicity in simian virus 40-transformed 3T3 cells transfected with ![]() |
15. | Haemmerli, G., and P. Strauli. 1981. In vitro motility of cells from human epidermoid carcinomas. A study by phase-contrast and reflection-contrast cinematography. Int. J. Cancer. 27: 603-610 |
16. | Harlow, E., and D. Lane. 1988. Preparing total cell lysates for immunoblotting. In Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 481 pp. |
17. |
Hemmings, L.K.,
P.A. Kuhlman, and
D.R. Critchley.
1992.
Analysis of the actin-binding domain of ![]() |
18. | Hirohashi, S., M. Watanabe, Y. Shimosato, and T. Sekine. 1984. Monoclonal antibody reactive with the sialyl-sugar residue of a high molecular weight glycoprotein in sera of cancer patients. Gann. 75: 485-488 |
19. | Hosaka, S., M. Suzuki, M. Goto, and H. Sato. 1978. Motility of rat ascites hepatoma cells, with reference to malignant characteristics in cancer metastasis. Gann. 69: 273-276 |
20. |
Hsu, D.K.W.,
Y. Gou,
G.F. Alberts,
K.A. Peifley, and
J.A. Winkles.
1996.
Fibroblast growth factor-1-inducible gene FR-17 encodes a nonmuscle ![]() |
21. |
Imamura, M.,
T. Sakurai,
Y. Ogawa,
T. Ishikawa,
K. Goto, and
T. Masaki.
1994.
Molecular cloning of low-Ca2+-sensitive-type nonmuscle ![]() |
22. | Kaplan, E.L., and P. Meier. 1958. Nonparametric estimation from incomplete observations. J. Am. Statis. Assoc. 53: 457-481 . |
23. | Klagsbrun, M.. 1996. The fibroblast growth factor family: structural and biological properties. Prog. Growth Factor Res. 167: 261-268 . |
24. |
Knudsen, K.A.,
A.P. Soler,
K.R. Johnson, and
M.J. Wheelock.
1995.
Interaction of ![]() ![]() |
25. |
Kuhlman, P.A.,
L. Hemmings, and
D.R. Critchley.
1992.
The identification and
characterisation of an actin-binding site in ![]() |
26. | Laemmli, U.K.. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680-685 |
27. | Lauffenburger, D.A., and A.F. Horwitz. 1996. Cell migration: a physically integrated molecular process. Cell. 84: 359-369 |
28. |
Lazarides, E.B., and
K. Burridge.
1975.
![]() |
29. | Matsudaira, P.. 1991. Modular organization of actin crosslinking proteins. Trends Biochem. Sci. 16: 87-92 |
30. |
Millake, D.B.,
A.D. Blanchard,
B. Patel, and
D.R. Critchley.
1989.
The cDNA
sequence of a human placental ![]() |
31. | Miyamoto, S., H. Teramoto, O.A. Coso, J.S. Gutkind, P.D. Burbelo, S.K. Akiyama, and K.M. Yamada. 1995. Integrin function: molecular hierarchies of cytoskeletal and signaling molecules. J. Cell Biol. 131: 791-805 [Abstract]. |
32. | Nicolson, G.L.. 1988. Cancer metastasis: tumor cell and host organ properties important in metastasis to specific secondary sites. Biochim. Biophys. Acta. 948: 175-224 |
33. | Nishihira, T., Y. Hashimoto, M. Katayama, S. Mori, and T. Kuroki. 1993. Molecular and cellular features of esophageal cancer cells. J. Cancer Res. Clin. Oncol. 119: 441-449 |
34. |
Otey, C.A.,
F.M. Pavalko, and
K. Burridge.
1990.
An interaction between ![]() ![]() |
35. |
Otey, C.A.,
G.B. Vasquez,
K. Burridge, and
B.W. Erickson.
1993.
Mapping of
the ![]() ![]() |
36. | Partin, A.W., J.S. Shoeniger, L.L. Mohler, and D.S. Coffey. 1989. Fourier analysis of cell motility: correlation of motility with metastatic potentials. Proc. Natl. Acad. Sci. USA. 86: 1254-1258 [Abstract]. |
37. | Prendergast, G.C., and E.B. Ziff. 1991. Mbh1: a novel gelsolin/severin-related protein which binds actin in vitro and exhibits nuclear localization in vivo. EMBO (Eur. Mol. Biol. Organ.) J. 10: 757-766 [Abstract]. |
38. | Raffioni, S., and R.A. Bradshaw. 1992. Activation of phosphatidylinositol 3-kinase by epidermal growth factor, basic fibroblast growth factor, and nerve growth factor in PC12 pheochromocytoma cells. Proc. Natl. Acad. Sci. USA. 89: 9121-9125 [Abstract]. |
39. | Rosen, P.P., and H.A. Oberman. 1992. Tumors of the mammary gland. Armed Forces Institute of Pathology, Washington, D.C. 115-175. |
40. |
Royal, I., and
M. Park.
1995.
Hepatocyte growth factor-induced scatter of Madin-Darby canine kidney cells requires phosphatidylinositol 3-kinase.
J. Biol.
Chem.
270:
27780-27787
|
41. | Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning. A Laboratory Manual, 2nd Ed. Cold Springs Harbor Laboratory Press, Cold Spring Harbor, NY. |
42. | Sato, Y., K. Mukai, S. Watanabe, M. Goto, and Y. Shimosato. 1986. The AMeX method: a simplified technique of tissue processing and paraffin embedding with improved preservation of antigens for immunostaining. Am. J. Pathol. 125: 431-435 [Abstract]. |
43. |
Shibata, T.,
A. Ochiai,
Y. Kanai,
S. Akimoto,
M. Gotoh,
N. Yasui,
R. Machinami, and
S. Hirohashi.
1996.
Dominant negative inhibition of the association between ![]() ![]() |
44. | Stossel, T.P.. 1993. On the crawling of animal cells. Science. 260: 1086-1094 |
45. | Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA. 76: 4350-4354 [Abstract]. |
46. | Vemuri, G.S.R. S.E.. 1994. Wortmannin inhibits serum-induced activation of phosphoinositide 3-kinase and proliferation of CHRF-288 cells. Biochem. Biophys. Res. Commun. 202: 1619-1623 |
47. |
Wehland, J.,
M. Osborn, and
K. Weber.
1979.
Cell to substratum contacts in living cells. A direct correlation between interference reflection and indirect
immunofluorescence microscopy using antibodies against actin and ![]() |
48. | Westley, B.R. H.. 1979. Estradiol induced proteins in the MCF7 human breast cancer cell line. Biochem. Biophys. Res. Commun. 90: 410-416 |
49. |
Witke, W.,
A. Hofmann,
B. Koppel,
M. Schleicher, and
A.A. Noegel.
1993.
The Ca(2+)-binding domains in nonmuscle type ![]() |
50. | Yamada, T., S. Hirohashi, Y. Shimosato, T. Kodama, S. Hayashi, T. Ogura, S. Gamou, and N. Shimizu. 1985. Giant cell carcinomas of the lung producing colony-stimulating factor in vitro and in vivo. Gann. 76: 967-976 |
51. | Yamada, T., J. Jiping, R. Endo, M. Gotoh, Y. Shimosato, and S. Hirohashi. 1995. Molecular cloning of a cell-surface glycoprotein that can potentially discriminate mesothelium from epithelium: its identification as vascular cell adhesion molecule 1. Br. J. Cancer. 71: 562-570 |
52. | Young, R.A., and R.W. Davis. 1983. Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. USA. 80: 1194-1198 [Abstract]. |
53. |
Youssoufian, H.,
M. McAfee, and
D.J. Kwiatkowski.
1990.
Cloning and chromosomal localization of the human cytoskeletal ![]() ![]() |
54. |
Zhang, L.,
M.C.P. Rees, and
R. Bicknell.
1995.
The isolation and long-term culture of normal human endometrial epithelium and stroma.
J. Cell Sci.
108:
323-331
|
55. | Zetter, B.R.. 1990. The cellular basis of site-specific tumor metastasis. N. Engl. J. Med. 322: 605-612 |