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© The Rockefeller University Press,
0021-9525/1998//1383 $5.00
The Journal of Cell Biology, Volume 140, Number 6,
, 1998 1383-1393
Article |
Actinin-4, a Novel Actin-bundling Protein Associated with Cell Motility and Cancer Invasion
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
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.
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.
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 β1 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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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.
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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 β-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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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.
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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.
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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.
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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.
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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.
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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 β1 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 β15, 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 β15 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.
Submitted: 21 July 1997
Revised: 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.
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