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Tumor Suppression & Functional Genomics Project, National Cancer Center Research Institute, Tokyo 104-0045 [M. Y., M. K., M. M., T. F., H. F., T. M., Y. M.], and Department of Genetics, Institute of Medical Science, University of Tokyo, Tokyo 108-0071 [M. Y., M. S.], Japan
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Introduction |
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40% of primary NSCLC tumors as well as in 20–30% of tumors from the liver, pancreas, and prostate (1
, 2)
. An extracellular domain of TSLC1 contains three immunoglobulin-like C2-type loops, suggesting the involvement of cell adhesion. A truncating mutation of TSLC1 corresponding to the cytoplasmic domain in a primary NSCLC suggests that this short domain of 47 amino acids is also important for tumor suppression. This cytoplasmic domain of TSLC1 contains a protein 4.1 binding motif and shows significant homology with that of human glycophorin C and Drosophila Neurexin IV. Glycophorin C associates with protein 4.1 and plays crucial roles in membrane deformity of erythrocytes (3)
, whereas Neurexin IV binds to Coracle, a Drosophila protein 4.1 homologue, and participates in the maintenance of the septate junction (4)
. The protein 4.1 family molecules present significant similarity with ezrin, radixin, and moesin (ERM subfamily) and merlin, a gene product of the tumor suppressor NF2, all of which are known to act as anchors for several transmembrane proteins to the actin cytoskeleton through their protein4.1/ezrin/radixin/moesin domain and the spectrin-actin binding domain (5
, 6)
. Therefore, it is reasonable to speculate that the activity of tumor suppression by TSLC1 would also require its anchoring to plasma membrane through a molecular apparatus similar to that seen in glycophorin C or Neurexin IV. The protein 4.1 family consists of four distinct molecules, including 4.1R, 4.1G, 4.1N, and DAL-1 (4.1B; Ref. 5
). Although the former three molecules are not reported to be involved in lung carcinogenesis, DAL-1 was shown to be down-regulated in considerable portions of primary lung tumors as well as lung cancer cell lines (7)
. Furthermore, restoration of DAL-1 expression to a normal level in NSCLC cell lines significantly suppressed cell growth in vitro, which suggests its role as a tumor suppressor (7)
. These reports led us to examine the possible interaction of TSLC1 and DAL-1, two distinct tumor suppressor proteins in human lung cancer. |
Materials and Methods |
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Expression Vectors, Antibodies, and Chemicals.
DAL-1 cDNA was provided from Dr. Takahiro Nagase (Kazusa DNA Research Institute) and cloned into a pcDNA3.1/V5-His TOPO TA vector to generate pcDNA3.1/V5-DAL-1. DAL-1-65 cDNA was amplified by reverse transcription-PCR using the human adult lung poly(A) RNA with the primers 5'-TAGCAGTAAACTCTCTCGGTTTTCA-3' and 5'-TCCACTGGACTCCGTCTTGGTTTCC-3' and subcloned into a pcDNA3.1/V5-His TOPO TA vector. For the construction of the expression vectors of GST-TSLC1 fusion proteins, three fragments corresponding to the COOH-terminal portions of TSLC1 were amplified by PCR from pcTSLC1 (1)
and subcloned into pGEX-4T-1 (Amersham Pharmacia). A synthetic polypeptide of 18 amino acids, N-INAEGGQNNSEEKKEYFI-C, at the carboxyl end of TSLC1 fused with KLH was used as an immunogen to raise rabbit anti-TSLC1 pAb CC2.
Immunoprecipitation and Western Blotting.
Twenty-four h after transfection with pcDNA3.1/V5-DAL-1, HEK293 cells were washed and treated with a lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and protease inhibitor mixture (Calbiochem)] on ice for 10 min and centrifuged, and protein content was determined using a protein assay reagent (Bio-Rad). One µg of the antibody was added to 500 µg of the cell lysate and incubated overnight at 4°C, then a 50% suspension of Protein A-Sepharose 6MB (Amersham Pharmacia) was added and incubated for 1 h at room temperature. Beads were then washed four times with a lysis buffer and resuspended in a NuPAGE sample buffer (Invitrogen). Immunoprecipitates or 5 µg of whole cell lysates were electrophoresed on 4–12% NuPAGE minigels (Invitrogen) and transferred to a polyvinylene difluoride membrane (Millipore) with a semi-dry transfer blot system (Bio-Rad). After blocking with Tris-buffered saline containing 1% Tween 20 and 5% skim milk for 30 min, the filters were incubated with primary antibody for 1 h, washed, and incubated with the appropriate horseradish peroxidase-labeled secondary antibody (Amersham Pharmacia). Specific proteins were detected using an enhanced chemiluminescence system (Lumi-LightPLUS; Roche).
GST Pull-Down Assay.
GST-TSLC1 fusion proteins expressed in Escherichia coli were purified using glutathione Sepharose 4B (Amersham Pharmacia). [35S]Methionine-labeled DAL-1-65 was synthesized in reticulocytes from pcDNA3.1/V5-DAL-1-65 using a TNT T7 Quick Coupled transcription/translation system (Promega). For in vitro binding, labeled proteins were incubated with equal amounts of GST-TSLC1 fusion proteins coupled with glutathione Sepharose beads for 1 h at 30°C in a NETN buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA (pH 8.0), 0.5% NP40, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin]. After incubation, the beads were washed with the NETN buffer and subjected to SDS-PAGE (10–20% ready gel; Bio-Rad). The gels were stained with Coomassie Brilliant Blue (ICN), and binding of 35S-labeled proteins was detected by autoradiography.
Immunofluorescence Microscopy Analysis.
HEK293 and U251 cells transfected with pcDNA3.1/V5-DAL-1-65 were selected against G418 for 2 weeks, and pooled clones were obtained. Cells were seeded on a Collagen I-coated culture slide (Biocoat; Becton Dickinson) and treated with 100 ng/ml of TPA (Sigma) at 30 min or 2 µM of cytochalasin D (Sigma) at 10 h. After being washed with PBS, cells were fixed with 4% paraformaldehyde for 15 min and then with 0.2% Triton X-100 in PBS for 5 min. Cells were subsequently incubated with a blocking solution (5% normal donkey serum, 0.02% sodium azide in PBS) for 1 h and incubated overnight with primary antibodies at 4°C. The cells were washed and incubated with FITC- or tetramethylrhodamine isothiocyanate-labeled secondary antibodies (Jackson Immunoresearch) in the presence of Alexa Fluor 633 dye-labeled phalloidin (Molecular Probes) for 1 h at room temperature. Samples were then washed and mounted in 80% glycerol and viewed with a laser-scanning confocal system (Radience 2000; Bio-Rad).
Northern Blot Analysis.
Poly(A) RNA was extracted from NSCLC cell lines using the FastTrack 2.0 kit (Invitrogen). One µg of poly(A) RNA was subjected in electrophoresis on 1% agarose-formaldehyde gels and transferred to Hybond-N+ (Amersham Pharmacia). A HindIII and XhoI fragment of 1651 bp from pcDNA3.1/V5-DAL-1-65 and a 961-bp PCR-derived fragment of TSLC1 cDNA (1)
were used as probes for the detection of DAL-1 and TSLC1, respectively, after labeling with [32P]dCTP by a Multiprime DNA labeling system (Amersham Pharmacia).
Examination for Metastasis from the Spleen to the Liver.
Three independent A549 cell lines stably expressed full-length TSLC1 (ATSLC1, ATSLC2, and ATSLC3), and the A549 cells carrying the control plasmid pcDNA3.1-Hygro(+) were obtained as described previously (1)
. The metastatic activity of the A549 cell and its derivatives in vivo were examined as reported previously (8)
. In brief, 5 x 105 cells on 0.05 ml of PBS were injected into the medial splenic tip of the anesthetized BALB/c nu/nu mice (Japan Crea Laboratory, Tokyo, Japan). Eight weeks after injection, the animals were sacrificed, and all of the visceral organs were examined for macroscopic metastasis as well as micrometastasis after fixation.
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Results and Discussion |
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, Neurexin IV, paranodin, syndecan-2, and glycophorin C also share this motif, and these proteins are shown to be directly associated with a member of protein 4.1 family molecules. This raised the possibility that TSLC1 might interact with DAL-1, a member of protein 4.1 family molecules. To analyze the possible association, we first generated a pAb, CC2, against the cytoplasmic tail of TSLC1, which detected a single molecule of exogenously expressed TSLC1 of Mr 75,000 in Cos-7 cells. A significant amount of endogenous TSLC1 of the same size has also been observed in a human embryonic kidney cell line, HEK293, and a glioblastoma cell line, U251 (Fig. 1b)
. When DAL-1 tagged with V5 was expressed in HEK293 cells and extracts of the cells were immunoprecipitated with anti-TSLC1 pAb, DAL-1 of Mr 110,000 was coprecipitated with endogenous TSLC1 and was detected by anti-V5 mAb (Fig. 1c)
. These findings suggest that TSLC1 directly associates with DAL-1 in vivo. Immunoprecipitation with anti-V5 mAb could not detect coprecipitated TSLC1 in the same cell lysates, probably because exogenously expressed DAL-1 was much more abundant than endogenous TSLC1.
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4.1) and a PDZ binding motif (TSLC1-CPDZ), respectively, were constructed in vitro as fusion proteins with GST (Fig. 1a)
. A GST pull-down assay was then carried out with [35S] methionine-labeled DAL-1-65, a central portion of DAL-1 containing an entire protein 4.1/ezrin/radixin/moesin domain, and a putative spectrin-actin binding domain. GST-TSLC1-C and GST-TSLC1-CPDZ significantly bound to DAL-1 almost at an equal level, whereas the binding activity of GST-TSLC1-C4.1 was reduced to the same level as that of GST alone, suggesting that TSLC1 binds to DAL-1 through its protein 4.1 binding motif (Fig. 1d)
.
Next, subcellular distribution of TSLC1 and DAL-1 was examined using immunofluorescence microscopy. HEK293 cells expressing DAL-1-65 tagged with V5 were stained with anti-TSLC1 pAb for TSLC1 (red), anti-V5 mAb for V5 (green), and phalloidin for actin (magenta). TSLC1 and DAL-1 colocalized along the cell membrane, displaying honeycomb-like patterns of staining when cells are cultured in confluence (Fig. 2, a–c)
. Actin filaments were also observed at the cell periphery in a similar pattern (Fig. 2d)
. When an early stage of cell adhesion was examined by culturing cells in low density, both TSLC1 and DAL-1 were localized primarily at the cell-cell attached sites (Fig. 2, f–h)
, whereas the actin cytoskeleton was organized along an entire plasma membrane, including the cell-cell attached sites (Fig. 2i)
. Taken together, these results suggest that TSLC1 interacts with the actin cytoskeleton through DAL-1.
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, cell-cell attachment was partly destroyed, and the cytoskeletal organization of actin was lost from the detached membrane (indicated by arrow). Corresponding to this change, both TSLC1 and DAL-1 also disappeared from the unattached membranes. On the other hand, TSLC1 remained associated with DAL-1 at the sites where cell-cell attachment was still intact (Fig. 2, k–m)
. These findings suggest that the colocalization of TSLC1 and DAL-1 is dependent on the integrity of the actin cytoskeleton.
To additionally investigate the role of TSLC1 and DAL1 in cytoskeletal organization, U251 cells expressing exogenous DAL-1 were treated with a phorbor ester, TPA, a potent activator of protein kinase C. TSLC1 and DAL-1 were colocalized at the cell periphery when U251 cells were cultured in low density without TPA (Fig. 3, a–c)
. Signals of TSLC1 were also detected in the nucleus, although their significance was unclear. Stress fibers of actin filaments were typically organized in U251 cells as seen in Fig. 3d
. When cells were treated with TPA, stress fibers were broken down, but membrane ruffling was induced by reorganization of the actin cytoskeleton (Fig. 3i)
where TSLC1 and DAL-1 were redistributed (Fig. 3, f–h)
. These results suggest that dynamic distribution of TSLC1 and DAL-1 to the actin cytoskeleton might be involved in not only cell adhesion but also cell motility. Staining of TSLC1 and DAL-1 was also detected at the perinuclear space with actin filaments, suggesting that the complex might be organized in the Golgi apparatus.
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. Three cell lines, ABC-1, RERF-LC-MS, and VMRC-LCD, expressed both TSLC1 and DAL-1. This finding is significant because Hirai et al. (8)
had reported that these cells showed reduced metastatic ability from the spleen to the liver in nude mice. Thus, to examine whether TSLC1 could be directly involved in the suppression of metastasis in NSCLC, three independent A549 derivatives, ATSLC1, ATSLC2, and ATSLC3, which stably expressed full-length TSLC1, as well as the A549 cells transfected with only plasmid DNA, were injected into the spleen in nude mice. Twelve of 16 mice injected with A549 cells carrying the control plasmid formed tumors in the liver, as determined when the mice were sacrificed 8 weeks after injection (Fig. 4c)
. In contrast, none of the 23 mice injected with ATSLC1, ATSLC2, or ATSLC3 cells developed tumors in the liver. No tumors were detected in the original sites of injection in the spleen or other visceral organs in either group of mice. These results indicate that TSLC1 has a strong suppressor activity of metastasis in vivo when expressed in TSLC1-deficient NSCLC cells. Taken together, loss of TSLC1 and DAL-1 function could be involved synergistically in lung cancer formation and/or metastasis.
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E-cadherin, another tumor suppressor of the transmembrane adhesion molecule, also interacts with the cytoskeleton and is involved in the invasion or metastasis of gastric and several other cancers. Numbers of cytoplasmic proteins, including 
- and ß-catenins and adenomatous polyposis coli, associate with E-cadherin directly or indirectly and provide targets for mutation in human cancer (11)
. Similarly, TSLC1 and DAL-1, acting in the same cascade, could be the targets for inactivation in human lung carcinogenesis. In fact, loss of expression of TSLC1 and DAL-1 frequently occurs in primary lung cancer and cell lines. Our findings, however, do not exclude the possibility that TSLC1 might also be associated with other members of protein 4.1 molecules. Additional studies would elucidate the possible interaction between TSLC1 and these molecules in various tissues. TSLC1 also harbors a PDZ binding motif at the cytoplasmic end in addition to the protein 4.1 binding motif, suggesting that it can associate with additional proteins. Finding more partners of TSLC1, as well as searching for their alterations in lung cancer, would provide a deeper background for understanding the significance of this pathway in lung tumor suppression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported, in part, by a Grant-in-Aid for the Second Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor, and Welfare of Japan; a Grant-in-Aid for Special Projects for Cancer Research from the Ministry of Education, Culture, Science, Sports, and Technology of Japan; and a grant from the Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research (OPSR) of Japan. M. M. is a recipient of research fellowships from OPSR, and M. K., H. F., and T. F. are recipients of research resident fellowships from the Foundation for Promotion of Cancer Research of Japan. 
2 To whom requests for reprints should be addressed, at Tumor Suppression & Functional Genomics Project, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. Phone: 81-3-3547-5295; Fax: 81-3-5565-9535; E-mail: ymurakam{at}gan2.ncc.go.jp
3 The abbreviations used are: NSCLC, non-small cell lung cancer; pAb, polyclonal antibody; mAb, monoclonal antibody; GST; glutathione S-transferase; TPA, 12-O-tetradecanoylphorbol-13-acetate.
Received 4/25/02. Accepted 7/31/02.
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