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[Cancer Research 63, 7979-7985, November 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

The Cytoplasmic Domain Is Critical to the Tumor Suppressor Activity of TSLC1 in Non-Small Cell Lung Cancer

Xinliang Mao1, Eric Seidlitz2, Kakoli Ghosh1, Yoshinori Murakami3 and Hara P. Ghosh1

1 Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada;
2 Hamilton Regional Cancer Center, Hamilton, Ontario, Canada;
3 Tumor Suppression and Functional Genomics Project, National Cancer Center Research Institute, Tokyo, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The tumor suppressor gene in lung cancer (TSLC1) encodes a membrane glycoprotein containing extensive homology in the extracellular domain with the immunoglobulin-superfamily cell adhesion molecules. The intracellular cytoplasmic domain (CT) contains a protein 4.1 (FERM) binding motif, and a PDZ-interacting motif. Expression of TSLC1 is silenced in non-small cell lung cancer and in other cancers by promoter hypermethylation. Restoration of TSLC1 expression suppresses tumorigenicity of lung cancer cells. We report here the critical role of the FERM-binding and PDZ- interacting domains of TSLC1 in tumor suppressor activity in non-small cell lung cancer. The entire CT domain [amino acid (aa) 398–442], the FERM binding motif (aa 398–410), or the PDZ-interacting motif (aa 432–442) was deleted to generate mutants CT1, CT3, and CT4, respectively. The lung cancer cell line A549, deficient in TSLC1 expression, was stably transfected with the wild-type TSLC1 or the deletion mutants. The cell lines were then injected into athymic (nu/nu) nude mice, and tumor formation at the sites of injection was monitored. A549 cells stably transfected with the empty vector or mutant TSLC1 constructs induced tumors at the sites of injection within 10 days. In contrast, A549 cells expressing wild-type TSLC1 showed the appearance of tumors after 35 days, and the tumors grew substantially slower. A549 cells expressing wild-type TSLC1 also showed suppression of anchorage-independent colony formation in soft agar and markedly increased cell-cell adhesion activity. These results suggest that the cytoplasmic domain of TSLC1 is important in its tumor suppressor activity, and the tumor suppression activity involve protein(s) interacting with the FERM- and PDZ-interacting regions.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TSLC1is a recently identified tumor suppressor gene involved in human lung cancer (1, 2, 3, 4) , breast cancer (5) , prostate cancer (6) , pancreatic cancer (7) , and other cancers (8) . TSLC1 is located at the region of 11q23.2 and spans >300 kb. Northern blot analysis of a number of tissues revealed that there are two TSLC1 transcripts of 4.4 kb and 1.6 kb, respectively, which have been shown to encode identical proteins. TSLC1 protein is a transmembrane glycoprotein of 442 amino acids consisting of an extracellular domain containing three immunoglobin-like C2-type domains, the transmembrane domain and the cytoplasmic domain (1 , 9) . The extracellular domain shows very high homology with those of other immunoglobulin superfamily CAMs,4 particularly with the neural CAMs, NCAM1 and NCAM2 (1 , 3) . Biochemical studies have shown recently that TSLC1 is involved in cell-cell adhesion (9) . A primary NSCLC tumor harboring a frameshift mutation of 2-bp deletion between the codon 443 and 444 suggested that the cytoplasmic domain may be important for tumor suppressor activity (1) . This domain harbors a protein FERM binding motif and a PDZ-interacting motif (1 , 10 , 11) . FERM domain-containing proteins have been known to be associated with tumorigenesis and link transmembrane proteins to actin cytoskeleton through their FERM domain and the spectrin-actin binding domain (12) . A direct association between TSLC1 and another tumor suppressor protein containing a FERM domain, DAL-1, has been established suggesting that DAL-1 interacts with TSLC1 through the FERM-binding region of TSLC1 (10) . PDZ domains are modular sequences that interact with specific COOH-terminal peptides of target proteins facilitating protein-protein interaction. PDZ domain-containing proteins have been shown to be involved in the assembly of multiprotein complexes, signal transduction, and subcellular targeting of proteins (13, 14) . Thus, the cytoplasmic domain of TSLC1 may interact with specific proteins via the FERM-binding and PDZ-interacting motifs, and may play a critical role in tumor suppression. TSLC1 is shown recently to interact directly with MPP3, one of the members of the MAGUK, through its PDZ-interacting motif (15) . However, the molecular function of TSLC1 protein in tumor suppression as well as the tumorigenic consequence of its loss of function is yet unknown. As a first step to understand the mechanism of TSLC1-induced tumor suppression, we examined the functions of the cytoplasmic domain, particularly, the FERM-binding and the PDZ-interacting motifs of TSLC1 in tumorigenesis in nude mice. The results show that deletion of these motifs abrogates tumor suppressor activity of TSLC1.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Mutants.
Wild-type full-length TSLC1 was cloned into a modified eukaryotic expression vector pcDNA3.1* (Hygro+; originally from Invitrogen, with the HindIII restriction site removed). The plasmids pc*TSLC1-CT1 (the entire cytoplasmic domain, aa 398–442 deleted), pc*TSLC1-CT3 (the FERM binding motif, aa 398–410 deleted), and pc*TSLC1-CT4 (PDZ binding motif, aa 432–442 deleted) were constructed by PCR amplification using pc*TSLC1 as templates and Pfu DNA polymerase (Stratagene). All of the mutants were sequenced before use.

Cell Line and Stable Transfection.
A549 cells were maintained in DMEM medium containing 10% FCS, 2 mM L-glutamine, 100 units/ml of penicillin, and 100 µg/ml of streptomycin (Life Technologies, Inc.). The day before transfection, A549 cells were split 1:5, the medium was changed the next day, and plasmid-DNA containing calcium phosphate precipitates were added to the cells. Four h later, cells were treated with a medium containing 15% glycerol for 2 min. Then the medium was replaced with complete medium, and cells were incubated for 2 days (16) . The cells transfected with various plasmids were then split 1:10, seeded into 12-well plates, and selected in the presence of 200 µg/ml of hygromycin (Roche) for about 10–15 days. The single colonies were isolated using cloning rings and expanded. The expressions of TSLC1and the mutant genes in the stably transfected cells were examined for RNA and protein levels by Northern and Western blotting, respectively. Six colonies of A549 cells, each stably expressing the wild-type or mutant TSLC1 genes, were isolated and characterized.

Northern Blotting Analysis.
Total RNAs from the stably transfected cells were isolated using TRIzol Reagent (Life Technologies, Inc.). Twenty µg of intact RNAs were denatured in a formaldehyde/formamide buffer, electrophoresed in a formaldehyde gel, and then transferred to nitrocellulose blotting membrane, BioTrace NT (PALL Co., Ann Arbor, MI) by the capillary method. To increase the binding of RNA to the membrane, the blots were subjected to UV cross-linking. TSLC1-specific DNA probe was prepared by labeling the 840-bp fragment (SacII to HindIII) with 32P (3) . The blot was washed in 0.1x SSC at 65°C for 60 min. The blot was reprobed with human glyceraldehyde-3-phosphate dehydrogenase cDNA as a control for loading.

Antibody Preparation and Purification.
An ectodomain fragment of TSLC1, aa 159–223, EC2, was cloned into the prokaryotic expression vector pGEX-2T (Amersham Pharmacia, Uppsala, Sweden) between the BamHI and EcoRI cloning sites. The fusion protein GST-EC2 was induced by isopropyl-1-thio-ß-D-galactopyranoside (BioShop Canada Co., Burlington, Ontario, Canada) and then purified using Glutathione Sepharose 4B (Amersham Pharmacia). It was additionally purified by fast protein liquid chromatography using Mono Q or Resource Q column (Amersham Pharmacia). GST-EC2 protein was used as an immunogen to raise rabbit anti-TSLC1 EC antibody. The anti-EC antibody was purified after the protocol proposed by Bar-Peled and Raikhel (17) . Briefly, crude serum was passed through a glutathione-agarose column and then through a GST-EC2-agarose column. The purified antibody was used for immunoblotting and immunofluroresence. An anti-CT antibody was also raised in rabbits by injecting a GST-fusion protein containing the COOH-terminal domain of TSLC1, aa 398–442. The anti-CT antibody was also purified as before.

Western Blotting Assay.
A549 cells stably transfected with pcDNA3.1* vector, pc*TSLC1, or with the mutants were washed twice with cold PBS, and then lysed with a prewarmed (85°C) gel loading buffer [50 mM Tris (pH 6.8), 100 mM DTT, 2% SDS, and 10% glycerol] and heated in a boiling water bath for 5 min. Samples were centrifuged after sonication. For tumor samples, tissues cut into very small pieces were suspended in ice cold radioimmunoprecipitation assay buffer [1% NP40, 0.4% sodium deoxycholic acid, 66 mM EDTA, 10 mM Tris (pH 7.5), 1 mM phenylmethylsulfonyl fluoride, and 1% Trasylol] and homogenized with a Polytron device (18) . The samples were then centrifuged, and the supernatant fractions were mixed with equal volume of 2 x gel loading buffer and heated in a boiling water bath. The samples (20 µg) were fractionated in 10% SDS-PAGE, and the separated proteins were transferred to a nitrocellulose membrane (BioTrace NT; PALL Co.). The membrane blot was incubated in 3% nonfat milk overnight at 4°C or 1 h at room temperature, and then with anti-EC antibody at 1:1,000 dilution for 1 h at room temperature. The blot was then incubated with the secondary antibody, peroxidase-conjugated AffiniPure F(ab')2 fragment antirabbit IgG (H+L; Jackson ImmunoResearch, PA) at 1:10,000 dilution for another hour. TSLC1 binding to the primary antibody was detected by reacting with Western Lighting Chemiluminescence reagent plus (Perkin-Elmer, Boston, MA), according to the manufacturer’s instruction.

Cell Growth in Soft Agar.
Anchorage-independent growth of the stably transfected cells were examined by colony formation in soft agar. Five x 103 or 1 x 104 cells were suspended in 0.3% agar and immediately overlayed on Petri dishes precast with 0.5% agar. The cells were incubated at 37°C. Two weeks later, colonies with a diameter >50 µm were counted (19) .

Cell Aggregation Assay.
Cell aggregation assays were performed as described previously (9 , 20) . Stably transfected A549 cells were split 1:2 and seeded in 100-mm plates 24 h before the experiment. The subconfluent cells were rinsed twice with Ca2+- and Mg2+-free PBS, detached by 0.2% trypsin, and dispersed by gentle pipetting. Then cells were washed once with Ca2+- and Mg2+-free HBSS (Life Technologies, Inc.), and were counted and resuspended as single cell suspensions containing 1 x 106 cells in Ca2+- and Mg2+-free HBSS, Ca2+- and Mg2+-containing HBSS, or Ca2+- and Mg2+-free HBSS plus 5 mM EDTA. The cell suspensions containing 1 x 106 cells were incubated in 15 ml polypropylene tubes at 37°C without shaking or reseeded in 12-well plates and incubated at 37°C on a gyratory shaker. Cell aliquots were taken out at time 20, 40, or 60 min, and the cell particles were counted under inverted microscope. Typical cell aggregates were also photographed.

Homodimerization Analysis.
Stably transfected A549 cells expressing wild-type TSLC1 or the mutants were incubated in PBS containing 3 mM BS3 (Pierce, Rockford, IL), at room temperature with constant agitation for 20 min. The reaction was quenched with the addition of 20 mM Tris-HCl (pH 7.5), and the cells were lysed in a buffer containing 10 mM iodoacetamide (Sigma), 1% NP40, and 0.5% Triton X-100. After clarification, the supernatants were subject to Western blot analysis (9) .

Subcellular Localization of COOH-Terminal Mutant TSLC1.
Wild-type and mutant TSLC1s were cloned into pEGFP-N3 vector (Clontech) to generate constructs pEGFP-TSLC1, pEGFP-TSLC1-CT1, pEGFP-TSLC1-CT3, and pEGFP-TSLC1-CT4. These four plasmids, or the empty vector pEGFP-N3, were transiently transfected into COS-1 cells, which were seeded on coverslips 1 day before the transfection. Twenty-four h later, cells were fixed with 2% paraformaldehyde at 4°C for 20 min. After drying, the coverslips were mounted on slides for analysis under a laser scanning confocal microscope (9) . Cells stably expressing TSLC1 or the mutants grown on coverslips were used for immunolocalization (1) . Briefly, cells were fixed in 2% paraformaldehyde for 15–20 min at 4°C, incubated with the primary antibody anti-EC at 4°C overnight, and then with the secondary antibody, fluorescein-conjugated goat antirabbit IgG antibody (ICN, Aurora, OH), at 37°C for 30 min. After washing and drying, the stained samples were mounted on slides with 50% glycerol. A confocal laser scanning microscope (Zeiss LSM 510) with a laser of argon at 488 nm was used for analysis and photomicrography.

Tumorigenesis in Nude Mice.
The functional analysis of cytoplasmic domains of TSLC1 was carried out in vivo as described previously (1) . A549 cells, stably transfected with the constructs pc*TSLC1, pc*TSLC1-CT1, pc*TSLC1-CT3, pc*TSLC1-CT4, or the empty-vector pcDNA3.1*, were prepared as single cell suspensions in PBS. Two x 105 cells in 0.2 ml PBS were injected into the two flanks of 5–6-week-old female BALB/C nu/nu athymic nude mice (Charles River). The tumor growth was monitored twice a week. The tumor volumes were calculated by the formula V = Length x Width x Height x 0.5 (19) . Four mice per group were used in each experiment, and the average tumor volumes were finally calculated. The experiment was repeated three times. All of the animal experiments were performed in accordance with the institutional guidelines.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
COOH-Terminal Mutants of TSLC1.
The COOH-terminal domain of TSLC1 shows the presence of two protein-protein interaction motifs. The sequence, 401RHKGTYFTHE410, forms the consensus FERM-binding sequence – RXK(X)0–4GXY(X)3E present in the cytoplasmic domains of glycophorin C, neurexin IV, paranodin, and syndecan (11 , 12) . The FERM-binding sequence present in TSLC1 is 85% identical to that of the human glycophorin C. Examination of the COOH-terminal amino acids of TSLC1 revealed that the sequence 439EFYI442 also forms the consensus class II PDZ-interacting sequence, -X{Phi}X{Phi}, present in glycophorin, neurexin, syndecan, and ephrin (11, 12, 13, 14) . To assess the importance of the FERM-binding and the PDZ-interacting motifs, as well as the COOH-terminal region of TSLC1, we constructed three deletion mutants (Fig. 1)Citation . The mutant TSLC1-CT1 was constructed by deleting residues 398–442 so that the entire COOH terminus was removed. The mutants, TSLC1-CT3 and TSLC1-CT4, lacking the FERM-binding domain or PDZ-interacting domain, were constructed by deleting residues 398–410 and 432–442, respectively.



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  		Fig. 1. Cytoplasmic domain mutants of TSLC1. The wild-type and COOH-terminal mutants were cloned into the eukaryotic expression vector pcDNA3.1*(Invitrogen) and then stably introduced into the human lung cancer cell line A549.

 
Mutants lacking the COOH terminus, FERM-binding, or PDZ-interacting motifs were then stably expressed in the NSCLC cell line A549, which showed a very low level of endogenous TSLC1 expression. A549 cells were transfected with pc*TSLC1, pc*TSLC1-CT1, pc*TSLC1-CT3, pc*TSLC1-CT4, and empty pcDNA3.1* vector, respectively. After 10–14 days of growth in the presence of hygromycin, 6 colonies from each transfected cell line were selected and expanded. The expressions of TSLC1 and its mutants were examined for both RNA and protein levels (Fig. 2)Citation . A549 cells as well as A549 cells transfected with pcDNA3.1* were found to express TSLC1-specific RNA and proteins at a very low level, whereas, the cells transfected with wild-type or mutant TSLC1 genes showed high levels of TSLC1-specific RNA and protein expression, indicating that the TSLC1 and its mutants were successfully introduced into the A549 cell lines. Previous reports (1 , 3) indicated that Northern blot analysis of a number of tissues revealed two TSLC1 transcripts of 4.4 kb and 1.6 kb. A549 cells stably transfected with TSLC1 or the mutants also expressed both 4.4-kb and 1.6-kb RNA (Fig. 2A)Citation . Analyses of the TSLC1-specific proteins by Western blot assay showed only one single band as detected by the TSLC1-specific anti-EC antibody (Fig. 2B)Citation . As expected, TSLC1 mutant CT1, which lacks the COOH-terminal 45 residues, moved slightly faster than the wild-type protein. Also, Western blot analyses using anti-CT antibody did not show the presence of the protein band corresponding to the CT1 mutant (data not shown). The sizes and the levels of the TSLC1-specific proteins expressed were comparable, indicating that the deletions in the COOH-terminal regions did not affect glycosylation or stability of the mutant proteins.



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  		Fig. 2. Identification of expressed RNA and proteins in A549 cells stably transfected with wild-type or mutant TSLC1genes. A, total RNAs were extracted from stably transfected or nontransfected A549 cells, and samples containing 20 µg total RNAs were analyzed by Northern blotting. The same blot was reprobed with human glyceraldehyde-3-phosphate dehydrogenase as a loading control (bottom). B, the wild-type and mutant TSLC1 proteins expressed in the cell lines were extracted in gel loading buffer and 20 µg samples analyzed by SDS-PAGE. The TSLC1 proteins were identified by immunodetection using anti-EC antibody (1:1000 dilution). The gels shown are representative of three experiments.

 
Subcellular Localization of TSLC1 and Its Mutants in A549 Cells.
Earlier we have shown that the protein TSLC1 is localized in the plasma and perinuclear membrane (1) . Deletion of an NH2-terminal segment containing the signal sequence led to the formation of a truncated protein, which failed to localize in the plasma membrane and showed no tumor suppressor activity, suggesting that cell surface localization may be critical for TSLC1 tumor suppressor activity (1) . Truncation of the COOH-terminal domain as well as the PDZ-interacting motif of a number of membrane proteins did not allow the mutant proteins to be localized in the cell surface and, thus, become biologically incompetent (16 , 21 , 22) . Therefore, we examined the cell surface localization of the mutant TSLC1 proteins. Wild-type and mutant TSLC1 proteins were fused to enhanced GFP, and the fusion proteins were expressed in COS-1 cells. The wild-type as well as the mutant GFP fusion proteins were localized in the cell surface (Fig. 3A)Citation . This was additionally confirmed by immunolocalization of the proteins in the stably transfected A549 cell lines, expressing wild-type or mutant proteins not tagged with GFP (Fig. 3B)Citation . Similar to the GFP fusion proteins, the mutants and the wild-type TSLC1 proteins were also localized in the plasma membrane. In confluent monolayers, the proteins were distributed along the cell surface showing a honeycomb-like pattern (Fig. 3B)Citation . Thus, deletion of the entire COOH-terminal region, the FERM-binding, or the PDZ-interacting motifs did not affect intracellular localization of the tumor suppressor TSLC1 protein.



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  		Fig. 3. Cell surface localization of wild-type or COOH-terminal mutants of TSLC1. A, wild-type and mutant TSLC1 proteins fused with GFP were expressed in COS-1 cells, and the GFP fluorescence was detected in a confocal microscope using a filter set suitable for fluorescein. B, wild-type and COOH-terminal mutants of TSLC1 stably transfected in A549 cells were detected by indirect immunofluorescence using anti-EC antibody and fluorescein-labeled goat antibody to rabbit IgG as the secondary antibody. Samples were analyzed with a confocal laser scanning microscope fitted with a filter set suitable for fluorescein. The labeling patterns shown are representative of minimum three to four separate experiments.

 
The Anchorage-Independent Growth in Soft Agar.
Generally, transformed cells have the ability of growing in an anchorage-independent manner, whereas nontransformed cells require anchorage to a substratum. Introduction of tumor suppressor genes in tumorigenic cells have resulted in the loss of anchorage-independent growth function (19 , 23 , 24) . Therefore, we assessed the importance of the COOH-terminal domain, the FERM-binding, or the PDZ-interacting motifs for the anchorage-independent growth function of the NSCLC A549 cells. Cells stably transfected with wild-type or mutant TSLC1 were seeded in soft agar medium and incubated at 37°C. In ~15 days well-isolated colonies were observed for all of the cells lines (Fig. 4A)Citation . Cells expressing wild-type TSLC1, however, formed colonies that were reduced both in the size and in the total number of colonies as compared with the size and number of colonies produced by A549 cells expressing the empty vector or the COOH-terminal mutants of TSLC1 (Fig. 4, A and B)Citation . Expression of TSLC1 reduced the ability of NSCLC A549 cells to grow in soft agar by 80%; additionally, the sizes of the colonies were reduced significantly. Thus, whereas 90% of the colonies produced by A549 cells expressing TSLC1 were ~50 µm or less in diameter, ~40% of the colonies produced by A549 cells expressing the empty vector were 75–100 µm in diameter. Cells expressing the deletion mutant showed a decrease in 30–50% in the number of colonies formed in soft agar. However, similar to A549 cells, these cells produced colonies larger in size than cells expressing wild-type TSLC1. Analyses of the size distribution showed that 25–40% of the colonies produced by A549 cells expressing mutant TSLC1 proteins were 75–100 µm in diameter (Fig. 4B)Citation . Taken together, the results indicated that expression of TSLC1 leads to a decrease in the ability of A549 cells for anchorage-independent growth.



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  		Fig. 4. Anchorage-independent growth of A549 cells expressing TSLC1 mutants. A, TSLC1 inhibited the cell growth in soft agar. The colonies from A549 cells expressing wild-type TSCL1 are smaller and fewer than those from A549 cells or those bearing TSLC1 mutants. B, size distribution of colonies formed in soft agar. Total colonies from 10 fields (x4) are presented. The data shown here are representative of three separate experiments.

 
Cell-Cell Adhesion Activity of TSLC1 Mutants.
TSLC1 shows a significant homology of the extracellular domain with those of CAMs belonging to immunoglobulin-CAM family, particularly with the neural cell adhesion proteins, N-CAM 1 and N-CAM 2, suggesting cell-cell adhesion activity of TSLC1 protein. Using a cell aggregation assay (20) , it was shown previously that TSLC1 mediates intracellular adhesion through homophilic trans-interaction (9) . Loss of cell-cell adhesion proteins has been recognized as one of the characteristics of transformed cells (25) . To determine whether the COOH-terminal region of TSLC1 is important in cell-cell adhesion activity of TSLC1, we, therefore, examined the effects of the COOH-terminal deletion mutants of TSLC1 on A549 cell aggregation. Single cell suspensions of A549 cells expressing wild-type or mutant TSLC1 were incubated in HBSS buffer in the presence or in the absence of Ca2+ and Mg2+, and the single cell particles were counted at various time points. A549 cells expressing the empty vector or the deletion mutants of TSLC1 did not form marked aggregates. In contrast, cells expressing TSLC1 formed aggregates (Fig. 5)Citation . Quantitation of the cell aggregation results showed that whereas ~76% of cells expressing wild-type TSLC1 formed aggregates, only 10–13% of cells expressing the empty vector or mutant TSLC1s could form aggregates under identical conditions (Table 1)Citation . The results suggest that the sequences present in the COOH-terminal region may be important in the cell-cell adhesion activity of TSLC1.



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  		Fig. 5. Cell adhesion activity of cells expressing TSLC1 or its mutants. A549 cells expressing TSLC1 form aggregates, whereas cells expressing TSLC1 mutants do not aggregate in HBSS with or without Ca2+/Mg2+. Shown here are the cell particles in the presence of Ca2+/Mg2+ after 60 min of incubation at 37°C with shaking. The results shown here are representative of four separate experiments.

 

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  		Table 1 TSLC1 induced aggregation in A549 cellsa

 
Dimerization of TSLC1 and Its Mutant Proteins on Plasma Membrane.
Masuda et al. (9) showed that wild-type TSLC1 protein forms a homodimer on the cell membrane in the nonpolarized MDCK cells and in HEK293 cells. Dimerization has been shown to be important for the biological functions of many proteins. To examine whether mutant TSLC1 proteins also form dimers, stably transfected A549 cells expressing wild-type or mutant TSLC1s were treated with 3 mM BS3, a membrane-impermeable cross-linker, for 20 min at room temperature (9) . Analyses of the cell lysates by Western blot using TSLC1-specific anti-EC antibody, a protein band of Mr ~160,000 size was detected in BS3-treated cells, whereas a protein band of Mr 80,000 size was observed in the extract of control cells not treated with BS3 (Fig. 6)Citation , indicating that both wild-type TSLC1 and its mutants formed homodimers on cell membrane. As expected the dimer of CT1 mutant protein moved slightly faster than the wild-type dimer. It may be noted that the amounts of homodimers formed were less than the monomers of TSLC1. It is possible that the anti-EC antibody does not recognize dimers as efficiently; the dimers are degraded rapidly or are not extracted as efficiently as monomers.



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  		Fig. 6. Formation of homodimers of TSLC1 and its mutant proteins. Cells expressing TSLC1 or its mutants were treated with a water-soluble cross-linker BS3 for 20 min, and the cell extracts were analyzed by Western blotting using the anti-EC antibody. The gels shown are representative of three experiments.

 
Tumorigenicity of A549 Cells Expressing Wild-Type or Mutant TSLC1.
We have demonstrated previously that expression of the tumor suppressor gene TSLC1 into the NSCLC A549 cells blocked tumorigenesis in nude mice (1) . To assess the effect of the COOH-terminal motifs on the tumor suppressor activity of TSLC1, we injected A549 cells stably expressing the empty plasmid vector, wild-type TSLC1, or the mutants into nude mice and examined the appearance of tumors at the site of injection. Mice injected with A549 cells bearing pcDNA3.1* vector developed visible tumors within 10 days, whereas A549 cells stably transfected with mutant TSLC1 genes formed tumors within 14 days. In contrast, mice injected with A549 cells expressing wild-type TSLC1 formed tumors after 35 days (Fig. 7B)Citation . Interestingly, although tumors started earlier by cells containing empty vector, tumors induced by cells expressing the mutants grew much faster. On the day of termination of the experiment, the average volume of the tumors from cells carrying TSLC1-CT3 and TSLC1-CT4 were twice those from the empty-vector carrying cells (Fig. 7, A and B)Citation . A549 cells expressing TSLC1 formed tumors at a much slower rate, and the average volumes of the tumors were about one fourth of that of the control mice injected with A549 cells transfected with empty vector. The tumors produced in the mice were analyzed for TSLC1-specific proteins, and the results showed that TSLC1 and its mutant proteins were well expressed in the tumors (Fig. 7C)Citation . Thus, the data show that the COOH-terminal motifs of TSLC1 are critical for its tumor suppressor activity.



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  		Fig. 7. Loss of tumor suppressor activity of the COOH-terminal mutants of TSLC1. Two x 105 A549 cells stably expressing wild-type or mutant TSLC1s were injected s.c. into the two flanks of 5–6-week-old female nude mice (BALB/c, nu/nu). A, tumors developed in the nude mice were photographed when the experiment was terminated at day 56 postinjection. B, tumor growth curve. Tumor volumes were calculated as described in "Materials and Methods." Results were reported as average for four mice in each group. C, analyses of TSLC1 and its mutant proteins from tumors produced in nude mice. Tumor tissues were extracted in radioimmunoprecipitation assay buffer by homogenization in a Polytron homogenizer. The supernatant proteins were mixed with equal volume of SDS-containing gel loading buffer, and 20 µg samples were analyzed by SDS-PAGE. TSLC1-specific proteins were detected by Western blot using anti-EC antibody (1:1000). The data presented here are representative of three separate experiments.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrates the importance of the cytoplasmic domain containing the FERM-binding and PDZ- interacting motifs in suppression of tumors in NSCLC by TSLC1. TSLC1 shows extensive sequence homology with immunoglobulin-CAM cell adhesion proteins in its extracellular domain (1) . Using a chemical cross-linker induced dimerization of TSLC1, it was shown previously that a homophilic cis-interaction may be involved in the cell adhesive function of TSLC1 (9) . Our results showed that the cytoplasmic domain was not required for the cis-homodimerization of TSLC1. Thus, the extracellular domain of TSLC1 may be sufficient for homophilic cis-interaction. Using a cell aggregation assay, it was also shown that TSLC1 mediated intracellular adhesion through homophilic trans-interaction, which was independent of divalent cations, Ca2+or Mg2+. TSLC1-deficient MDCK cells failed to form aggregates, whereas introduction of TSLC1 allowed MDCK cells to form aggregates independent of the presence of Ca2+or Mg2+ (9) . Our studies demonstrated that the NSCLC cell line A549 did not form cell aggregates in the presence or absence of divalent cations, but expression of TSLC1 induced cell aggregation. However, A549 cells expressing the mutant TSLC1 genes could not form cell aggregates, suggesting that the cytoplasmic domain may play an important role in the cell-cell adhesion activity of TSLC1. Results of colony formation in soft agar showed that the FERM-binding motif and the PDZ-interacting motif present in the cytoplasmic domain of TSLC1 may be essential for cell proliferation and cell adhesion. Studies with the Nf2 tumor suppressor gene have shown that mutations in the FERM domain also affected cell adhesion including anchorage-dependent growth as well as the tumor suppressor activity (24) .

Results of in vivo tumorigenesis in nude mice showed that deletion of the cytoplasmic domain, the PDZ-interacting motif, or the FERM-binding motif abrogated the tumor suppressor activity of TSLC1. Expression of TSLC1 in A549 cells not only reduced the growth of tumors at the site of injection but also prolonged the latency period significantly. In contrast, truncation of the cytoplasmic domain, and deletion of the FERM-binding or PDZ-interacting motifs of TSLC1 increased considerably the growth rate of the tumors. The enhanced growth rate of tumors induced by the deletion mutants suggests that these mutants could function in a dominant-negative manner. Mutant TSLC1 could possibly inactivate the very small amount of wild-type TSLC1 expressed in A549 cells through heterodimer formation and, thus, inactivate the function of the wild-type protein. Mutant versions of proteins including tumor suppressors are known to inactivate the wild-type function by a dominant-negative mechanism (26 , 27) . Truncation of the tumor suppressor gene ST7 was shown to abrogate its tumor suppressor function by dominant-negative inhibition (28) . Mutations in the p53 gene were also shown to inhibit wild-type p53 function by a dominant-negative mechanism (29 , 30) . Mutation in the FERM domain of NF2 was also reported to form a dominant-negative protein, which blocked the suppressor activity of the wild-type NF2 (24) . In the case of endometrial serous carcinoma, dominant-negative mutations of p53 were also suggested to play an important role of p53 inactivation in tumorigenesis (30) . Dominant-negative mutants are also known to induce a gain of tumor promotion function (31) . Thus, the TSLC1 mutants lacking the FERM-binding or the PDZ-interacting motifs may not only function in a dominant-negative fashion but also may induce a gain-of-function to enhance tumorigenicity of the A549 cells. The putative dominant-negative or the gain-in-function effects of the TSLC1 mutants could be tested by blocking the endogenous low expression of TSLC1 in A549 cells using stable suppression of gene expression by RNA interference.

In summary, our results demonstrate that the FERM-binding and PDZ-interacting motifs present in the cytoplasmic domain are critical for tumor suppressor activity in vivo, cell-cell adhesion, and proliferation in an anchorage-dependent manner. The FERM-binding motif of TSLC1 associates with DAL-1, another lung cancer tumor suppressor protein that links to the cytoskeletal actin filaments through its cytoplasmic domain (9 , 32) . However, other FERM-domain containing proteins may also act as binding partners, because reintroduction of only the TSLC1 gene into A549 cells, which lack both TSLC1 and DAL-1 proteins, was sufficient for tumor suppressor activity (9 , 32) . The PDZ-interacting motif was shown recently to interact with MPP3, one of the human homologues of a Drosophila tumor suppressor Dlg (15 , 33) . MPP3 (Dlg3) protein shows amino acid identity and similar domain structure to the erythroid protein p55, the prototypic member belonging to a subfamily of MAGUK proteins (33 , 34) . Comparison of the sequence of the COOH terminus of human erythrocyte transmembrane glycoprotein glycophorin C with that of TSLC1 shows that their cytoplasmic domain shows a 51% identity, whereas the FERM-binding and the PDZ-interacting motifs are 85% and 100% identical, respectively (1 , 10 , 11) . The FERM-binding motif of glycophorin C binds to the FERM domain of the erythrocyte 4.1 protein, which also contain an actin-spectrin binding domain acting as a membrane-cytoskeleton linker (34) . The COOH-terminal PDZ-interacting motif of glycophorin C was also shown to interact with the MAGUK erythrocyte protein p55 (MPP1), a homologue of the Drosophila discs-large tumor suppressor protein (35) . The p55 protein also interacted with 4.1 protein via its FERM-binding motif leading to the formation of a ternary complex between p55, glycophorin C, and protein 4.1, which is critical for membrane-cytoskeleton interaction and the shape of erythrocytes (34 , 35) . In view of the high level of identity between these two domains of TSLC1 and glycophorin C, it can be postulated that a ternary complex containing TSLC1, DAL-1, and MPP3 linked to the cytoskeleton may play a critical role in cell proliferation, cell-cell adhesion, and suppression of tumorigenesis.


   ACKNOWLEDGMENTS
 
We thank Drs. Andy Fletch, Kathy Delaney and Gurmit Singh for their support, and Dr. John Hassell for critical review of the article.


   FOOTNOTES
 
Grant support: National Cancer Institute, Canada.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Request for reprints: Hara P. Ghosh, Department of Biochemistry, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada, L8N 3Z5. Phone: (905) 525-9140 extension 22451; Fax: (905) 522-9033; E-mail: ghosh{at}mcmaster.ca

4 The abbreviations used are: CAM, cell adhesion molecule; CT, cytoplasmic domain; FERM, 4.1/ezrin/radixin/moesin; MAGUK, membrane-associated guanylate kinase; aa, amino acid; GST, glutathione S-transferase; BS3, bis(sulfosuccinimidyl) suberate; NSCLC, non-small cell lung cancer; GFP, green fluorescent protein. Back

Received 7/ 3/03. Revised 8/20/03. Accepted 8/26/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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