Expression of Tetraspanins in Human Lung Cancer Cells: Frequent Downregulation of CD9 and Its Contribution to Cell Motility in Small Cell Lung Cancer

Expression of tetraspanins in human lung cancer cells: frequent downregulation of CD9 and its contribution to cell motility in small cell lung cancer

Toshiki Funakoshi1, Isao Tachibana*,1, Yoshihiko Hoshida2, Hiromi Kimura1, Yoshito Takeda1, Takashi Kijima3, Kazumi Nishino1, Hiroyuki Goto1, Tsutomu Yoneda1, Toru Kumagai1, Tadashi Osaki1, Seiji Hayashi1, Katsuyuki Aozasa2 and Ichiro Kawase1

1Department of Molecular Medicine, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Sutia, Osaka 565-0871, Japan; 2Department of Pathology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Sutia, Osaka 565-0871, Japan; 3Division of Thoracic and Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA

Small cell lung cancer (SCLC) invades locally and lungcancer (SCLC) and nonsmall cell lungcancer metastasizes distantly extremely early when compared (NSCLC). SCLC is characterized by several neuroendo- with nonsmall cell lung cancer (NSCLC). The underlying crine features, as evidenced by the presence of dense core molecular mechanisms, however, have not been elucidated. granules, high enzymatic activities of L-dopa decarbox- Accumulating evidence suggests that downregulation of ylase, production of hormones and neuropeptides, and several members of tetraspanins is associated with expression of neural cell adhesion molecule (N-CAM) progression of solid tumors, thus indicating poor prog- (Carney et al., 1985). Clinically, SCLC is distinct from nosis. Here we screened 30 lung cancer cell lines for NSCLC in that most patients are inoperable at expression of tetraspanins, CD9, CD63, CD81, CD82, diagnosis, because the tumor locally invades and CD151, and NAG-2. Flow cytometry revealed that, distantly metastasizes extremely early (Mountain, among these proteins, CD9 is broadly expressed in 1978). The underlyingmolecular mechanisms of this NSCLC lines, but is absent or highly reduced in most early progression, however, are not clarified. SCLC lines (Po0.0001). Using the Boyden chamber and Tetraspanin, also called tetraspan or transmembrane- videomicroscopic cell motility assays, we showed that 4 superfamily (TM4SF), comprises more than 20 cell- stable transfection of CD9 into an SCLC line, OS3-R5, surface proteins includingCD9, CD37, CD53, CD63, reduced cell motility on fibronectin. Furthermore, by CD81, CD82, and CD151. All of these molecules share a transient transfection of green fluorescent protein (GFP)- characteristic structure that spans the membrane four tagged CD9 into three other SCLC lines, we observed that times thereby formingtwo extracellular loops. Although SCLC cells expressing GFP-CD9 were uniformly less the precise biological functions of tetraspanins remain motile than untransfected cells. CD9 or GFP-CD9 was elusive, experiments usingmonoclonal antibodies, associated with b1 integrins and distributed at the tumor cDNAs, and knockout mice have suggested its involve- cell periphery and cell–cell contacts, suggesting that CD9 ment in cell proliferation, activation, motility, and modifies b1 integrin function to reduce motility. These fusion (Hemler et al., 1996; Maecker et al., 1997; findings suggest that low expression of CD9 may Tachibana and Hemler, 1999; Le Naour et al., 2000; contribute to the highly invasive and metastatic phenotype Miyado et al., 2000). Based on coimmunoprecipitation of SCLC. and immunofluorescence results, tetraspanins are be- Oncogene (2003) 22, 674–687. doi:10.1038/sj.onc.1206106 lieved to function as molecular facilitators. This family of proteins connects other surface molecules including Keywords: small cell lungcancer (SCLC); tetraspanin; CD4, CD8, CD19, CD21, major histocompatibility CD9; motility complex (MHC) class I and II, and integrins as well as cytoplasmic signaling molecules such as phosphatidyli- nositol 4-kinase and protein kinase C into a tetraspan web (Rubinstein et al., 1996; Maecker et al., 1997; Introduction Hemler, 1998). Amongtetraspanins, CD9, CD63, and CD82 were Lungcancer has become the most common fatal tumor considered to be metastasis-inhibitory factors. Transfec- in the developed countries. It is classified into small cell tion of these molecules into certain tumor cells inhibited in vitro cell motility and in vivo tumor metastasis *Correspondence: I Tachibana; E-mail: [email protected] (Hemler et al., 1996). In addition, clinical and patholo- Received 18 October 2001; revised 30 September 2002; accepted 4 gical findings suggest that downregulation of CD9, October 2002 CD63, or CD82 is associated with the progression of Tetraspanins in human lung cancer cells T Funakoshi et al 675 solid tumors. Reduced expression of these molecules is 30 lungcancer lines is shown in Table 1. CD63, CD81, more frequently observed in metastatic tumors than CD82, and CD151 were broadly expressed in both primary sites, and patients with tumors lackingthese SCLC and NSCLC lines. NAG-2 was clearly positive in molecules tend to be at advanced stages and thus show several NSCLC lines, while it was absent or only poor survival. Downregulation in CD9 or CD82 is marginally stained in most SCLC lines (Po0.01). associated with the progression of differentiated carci- Remarkably, whereas all NSCLC lines but VMRC- nomas such as breast (Miyake et al., 1995, 1996; Huang LCD were CD9-positive (12 of 13), only a minor et al., 1998; Yang et al., 2000), pancreas (Guo et al., population of SCLC lines expressed CD9 (three of 17) 1996; Sho et al., 1998), colon (Cajot et al., 1997; (Po0.0001). We also stained these lungcancer lines with Lombardi et al., 1999), esophagus (Uchida et al., 1999), anti-MHC class I monoclonal antibody (MoAb). Con- and NSCLC (Higashiyama et al., 1995; Adachi et al., sistent with a previous report (Doyle et al., 1985), 1996, 1998), whereas CD63 reduction associated with expression of this molecule was less frequent in SCLC that of melanoma (Atkinson et al., 1985; Hotta et al., than in NSCLC (Po0.05). All CD9-positive SCLC lines 1988). Intriguingly, CD63 expression had no correlation also expressed class I molecules. with the spread of NSCLC and pancreatic cancer To clarify the mechanisms for CD9 downregulation, (Adachi et al., 1998; Sho et al., 1998). Recently, the eight CD9-negative SCLC lines were randomly selected loss of another tetraspanin, CD81, was reported to and CD9 gene expression was evaluated by reverse associate with differentiation and metastasis of hepato- transcription–polymerase chain reaction (RT–PCR). As cellular carcinomas (Inoue et al., 2001). shown in Figure 2, SCLC lines except OS2-RA Accumulating clinical and pathological findings have displayed absent or reduced levels of CD9 in contrast indicated coincidence of the downregulation of tetra- to abundant expression in NSCLC lines. OS2-RA spanins with the progression of differentiated carcino- appeared to have a moderate level of full-length CD9 mas. However, in vitro studies that account for these mRNA despite no reactivity against anti-CD9 MoAb findings still remain to be done. Moreover, little (Figure 1 and Table 1). We also examined CD63 gene information on tetraspanins has been obtained from expression and observed no difference between SCLC less differentiated yet more malignant cases such as and NSCLC. Together, among the six tetraspanins, SCLC. In the present study, we examined the expres- CD9 showed the highly biased expression between sions of six tetraspanins, which are ubiquitously SCLC and NSCLC, and the CD9 reduction in SCLC expressed in most organs including the lung, in 30 lung lines primarily occurs at or prior to the transcription cancer cell lines. Amongthese tetraspanins, CD9 level. A possibility, however, could not be excluded that showed highly biased expression between SCLC and the reduced levels of CD9 may be because of the NSCLC. Most SCLC lines expressed much lower levels instability of CD9 mRNA in SCLC lines. of CD9 than NSCLC lines. We further showed that CD9 expression into SCLC lines suppressed cell motility Ectopic expression of CD9 into OS3-R5 reduces cell on fibronectin (FN). motility on FN To investigate whether ectopic expression of CD9 alters SCLC cell behavior, we selected OS3-R5 and established Results OS3-R5-pZCD9, a CD9-overexpressingstable transfec- Expression of tetraspanins in lung cancer cell lines tant of OS3-R5. Successful CD9 introduction was confirmed by flow cytometry (Figure 3a) and immuno- Since little information has been reported on tetraspanin blotting(Figure 4a, left panel). Expressions of another expression of lungcancer lines, we first examined the tetraspanin, CD81, and b1 integrin of OS3-R5-pZCD9 expressions of CD9, CD63, CD81, CD82, CD151, and were comparable to that of a mock-transfectant (OS3- NAG-2 in SCLC lines and compared with those in R5-pZSV) or the parent (Figure 3a). We observed no NSCLC lines. Since a portion of SCLC lines might have significant difference in the in vitro proliferation rates lost neuroendocrine features and acquired NSCLC-like amongthese three lines (data not shown). In addition, phenotypes duringculture (Mabry et al., 1991), we these three lines displayed similar strongadherence to excluded such variant SCLC lines from this study by FN, but not to bovine serum albumin (BSA) or laminin testingfor the expression of N-CAM (see Materials and (LM) (Figure 3b). Next, we investigated whether CD9 methods), which is known as a cluster 1 SCLC antigen expression affects cell motility of OS3-R5 on FN. In a and is a differential marker between SCLC and NSCLC conventional Boyden chamber assay, a substantial (Souhami et al., 1987). Consequently, all SCLC lines number of parent cells and mock-transfectants transmi- and NSCLC lines tested in this study (Table 1) are N- grated through the FN-coated membrane into lower CAM-positive and N-CAM-negative, respectively. Flow chambers (Figure 3c). This motility is b1 integrin- cytometry of representative cell lines are shown in dependent because it was almost completely abolished Figure 1. Three NSCLC lines, A549, EBC-1, and Lu99, by anti-b1 integrin MoAb but not affected by control expressed CD9 and CD63 but not N-CAM. On the IgG or anti-MHC class I MoAb, and few cells other hand, three SCLC lines, OS3-R5, OS2-RA, and transmigrated through the uncoated or poly-l-lysine CADO LC6, expressed N-CAM and CD63 but not (PLL)-coated membranes. Remarkably, transmigration CD9. Summary of the expressions of six tetraspanins in of OS3-R5-pZCD9 cells was reduced to only 20% of

Oncogene Tetraspanins in human lung cancer cells T Funakoshi et al 676 Table 1 Expression of tetraspanins in LC cell lines Cell lines Histology Morphology Class I CD9 CD63 CD81 CD82 CD151 NAG-2 SCLC NCI-H69 Sma M/Fb +c À ++++++7 NCI-N231 Sm F + À ++ ++ ++ ++ À NCI-N857 Sm F 7 À ++ ++ + ++ À SBC-1 Sm F ÀÀ +++7 + À CADO LC6 Sm M + À ++ ++ + ++ À OC10 Sm F ++ À ++ ++ + ++ 7 OS1 Sm F ++ ++ + ++ ++ ++ + OS2-RA Sm M ÀÀ ++ ++ + ++ 7 OS3-R5 Sm M ++ À ++ ++ + + À OS4 Sm F + + ++ ++ + + 7 OS5 Sm M + À ++À 7 À OS6 Sm M/F ÀÀ ++À + À OS7 Sm F ++ À ++++ À OS10 Sm F ++ + + ++ + ++ 7 OS15 Sm F + À ++7 + À OS17 Sm F ÀÀ ++ ++ + ++ 7 OS18A Sm M + À ++ ++ + ++ À

NSCLC A549 Ad M ++ ++ + ++ À ++ + OAD2 Ad M ++ ++ ++ + ++ ++ 7 CADO LC9 Ad M/F ++ ++ À ++ À + À RERF-LC-MS Ad M ++ ++ ++ + + ++ + PC-3 Ad M ++ ++ À +++ À PC9 Ad M/F ++ + + + À + À VMRC-LCD Ad M ++ À ++ ++ 7 ++ À QG56 Sq M ++ ++ + ++ ++ ++ À OSQ1 Sq M ++ ++ + ++ 7 ++ À EBC-1 Sq M ++ ++ ++ ++ + ++ + HARA Sq M ++ ++ ++ ++ ++ ++ + Lu65 La M/F ++ ++ ++ + + + + Lu99 La M ++ ++ ++ ++ + ++ ++

P-valued Po0.05 Po0.0001 NSe NS NS NS Po0.01

aSm: small cell carcinoma; Ad: adenocarcinoma; Sq: squamous cell carcinoma; La: large cell carcinoma bLC cell lines grew as monolayer cultures (M), floating aggregates (F), or a mixture (M/F) cClass I and tetraspanin expressions were evaluated by flow cytometry. Stainingwas scored as ‘ À’, ‘7’, ‘+’, or ‘++’ if fluorescence intensity was otwofold, 2–3-fold, 3–10-fold, or >10-fold higher than staining observed for the control mouse IgG, respectively. Two additional experiments showed similar results. For CD9 expression, two MoAbs (MM2/57 and BU16) gave similar results dDifferences in the number of positive (+ or ++) lines between SCLC and NSCLC were analysed by the w2 test eNS, not significant

that of the mock transfectant or the parent. We further cytometry (data not shown), CD9 coprecipitated with studied the motility of OS3-R5-pZCD9 usinga time- anti-b1 integrin and anti-CD81 MoAbs but not with lapse videomicroscopic motility assay and obtained control or anti-N-CAM MoAbs. Thus, CD9 specifically similar results. The parent and the mock transfectant associates with b1 integrins but not with N-CAM in randomly migrated on FN, while the motility of OS3- OS3-R5-pZCD9. R5-pZCD9 cells was again reduced to 20% of the parent. All these three lines were static on PLL Transfection of green fluorescence protein (GFP)-tagged (Figure 3d). These data suggested that CD9 introduction CD9 into OS3-R5 suppresses b1 integrin-dependent motility of OS3-R5. To exclude the possibility that the reduction of OS3-R5- CD9 associates with b1 integrins but not with N-CAM pZCD9 cell motility resulted from clonal selection, we planned to again analyse CD9 effect by transient SCLC reportedly expresses adhesion proteins, b1 transfection with GFP-tagged CD9 (GFP-CD9) cDNA integrins and N-CAM, and both of these molecules (see below). Before transient transfection, we established are likely to regulate motility and metastasis of SCLC a stable OS3-R5 transfectant that expressed GFP-CD9 (Scheidegger et al., 1994; Bredin et al., 1998). To explore (OS3-R5-GFP-CD9) to confirm successful introduction the mechanisms involved in the reduction of OS3-R5- of GFP-CD9. Immunoblottingagainst both GFP and pZCD9 motility, coimmunoprecipitation experiments CD9 clearly showed the prominent existence of 50-kDa usingMoAbs against these molecules were performed GFP-CD9 protein in OS3-R5-GFP-CD9 lysate (Figure 4b, left panel). Although N-CAM expression (Figure 4a). Minor additional bands were also stained was more intense than b1 integrins or CD81 in flow and these were presumably degraded proteins. On the

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Figure 1 Expression of CD9, CD63, and N-CAM in SCLC and NSCLC lines. Tumor cells were stained with MM2/57 (anti-CD9), 6H1 (anti-CD63), or ITK-2 (anti-N-CAM), and then labeled with FITC-conjugated goat anti-mouse immunoglobulin (open histograms). Normal mouse IgG was used as a control (ﬁlled histograms). Stained cells were analysed on a FACSort

Figure 2 Reduced CD9 gene expression in SCLC lines. Total RNA was extracted from SCLC and NSCLC lines. A measure of 1 mgof the total RNA was reverse-transcribed and subjected to 25 PCR amplification cycles each consistingof 40 s at 94 1C, 40 s at 601C, and 90 s at 721C. The amplified DNA samples were electrophoresed on 1% agarose gels, and the bands were visualized with ethidium bromide. Primers used for CD9 (Higashiyama et al., 1995; Miyake et al., 1995) and CD63 (Adachi et al., 1998) have been previously described. b-actin cDNA amplification was used as the internal control other hand, a control line stably transfected with GFP To investigate the intracellular distribution of GFP- cDNA alone (OS3-R5-GFP) showed a single 27-kDa CD9, OS3-R5 cells were transiently transfected with GFP band that was not stained with anti-CD9 MoAb. GFP or GFP-CD9, and fluorescent cells were analysed We further confirmed cell-surface expression of GFP- (Figure 5a). Consistent with the previously reported CD9 usinganti-CD9 MoAb by flow cytometry and by distribution of CD9 in fixed and permeabilized cells immunoprecipitation from surface-biotinylated OS3- (Nakamura et al., 1995; Berditchevski and Odintsova, R5-GFP-CD9 (data not shown). We next investigated 1999), GFP-CD9 was distributed at the cell periphery if the GFP-CD9 protein associates with b1 integrins as and at cell–cell contacts in viable transfected cells. This well as untagged CD9 (Figure 4b, right panel). As was in clear contrast to the homogenous distribution of expected, the chimeric GFP-CD9 coprecipitated with b1 GFP. Despite its presence at cell–cell contacts, GFP- integrins and CD81 but not with N-CAM. Several CD9 overexpression did not appear to promote the degraded proteins also appeared to coprecipitate with b1 aggregation of tumor cells (Figure 5a). We further integrins and CD81. Similar results were obtained when carried out detailed analysis of the distribution of cell lysates of transient transfection were used (data not GFP-CD9 and b1 integrins after fixation and permea- shown). bilization. Although these treatments might remove

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Figure 3 CD9 expression in OS3-R5 reduces cell motility on FN. (a) Flow cytometry of OS3-R-5, a mock transfectant, OS3-R5- pZSV, and a CD9-overexpressingtransfectant, OS3-R5-pZCD9. These cells were stained with anti-CD9 MoAb, BU16, anti-CD81 MoAb, M38, anti-b1 integrin MoAb, SG/19, or mouse control IgG, labeled with FITC-conjugated goat anti-mouse immunoglobulin, and then analysed on a FACSort. (b) Adhesion assay of OS3-R5, OS3-R5-pZSV, and OS3-R5-pZCD9. Cells (1.5 Â 104) were allowed to adhere to the FN-, LM-, or BSA-coated wells. Unattached cells were washed away and then attached cells were quantiﬁed usingan MTT assay. Values are shown as percentages of the absorbance obtained from unwashed cells on PLL. (c) Boyden chamber motility assay of OS3-R5, OS3-R5-pZSV, and OS3-R5-pZCD9. Cells (1.5 Â 104) were applied to the upper chamber of Transwells that were uncoated or precoated with FN or PLL. Cells migrating through the membrane to the lower chamber were counted. Motility of the parent OS3-R-5 cells was also evaluated in the presence of 10 mg/ml of control IgG (*), anti-MHC class I MoAb, IOT2 (**), or anti-b1 integrin MoAb, SG/19 (***). For (b) and (c) each bar represents the mean and standard deviation of determinations made on triplicate cultures run in parallel. (d) Time-lapse videomicroscopic cell motility assay of OS3-R5, OS3-R5-pZSV, and OS3-R5-pZCD9. One thousand cells were plated on FN- or PLL-coated wells and monitored usingan inverted microscope equipped with a video recorder. Cell motility was traced and analysed with an imaging device, MCID, using the program Image Analyzer. The tracks and distances of random motility of at least 30 cells were determined for each cell line. Each bar represents the mean and standard deviation

substantial amounts of GFP-CD9 from the cell surface Transfection of GFP-CD9 into other SCLC lines (Berditchevski et al., 1997), Figure 5b nonetheless uniformly reduces cell motility on FN showed colocalization of GFP-CD9 and b1 integrins at the cell periphery and the cell–cell contact. These To confirm the motility–inhibitory effect of CD9 on findings suggested that the introduced GFP-CD9 SCLC, we performed transient transfection of GFP- protein was targeted to the membrane properly, and it CD9 into multiple SCLC lines includingOS3-R5. We would function to inhibit motility of SCLC cells in the selected three other CD9-negative SCLC lines that are same way as untagged CD9. To confirm this, we adherent to FN. Cell motility of these lines was performed a Boyden chamber motility assay using compared with NSCLC lines by the time-lapse video- OS3-R5 and its stable transfectants, OS3-R5-GFP and microscopic motility assay (Figure 6a). Notably, these OS3-R5-GFP-CD9. The transmigration through the SCLC cell lines includingOS3-R5 were more motile on FN-coated membrane of OS3-R5-GFP-CD9 was re- FN than NSCLC lines. All the SCLC and NSCLC cells duced to the similar level to OS3-R5-CD9, while that of were static on PLL, suggesting that their motility on OS3-R5-GFP was comparable to the parent, OS3-R5. FN was mediated by integrins. In Figure 6b, OS3-R5 None of these cell lines showed significant migration cells were transiently transfected with GFP or GFP- through the uncoated or PLL-coated membranes (data CD9, and motility of fluorescent cells was analysed. not shown). When cells were traced and their tracks were visualized,

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Figure 3 Continued

OS3-R5 cells expressingGFP alone are as motile as (data not shown). We also performed transfection of untransfected cells, whereas cells expressingGFP-CD9 GFP-tagged NAG-2 (GFP-NAG-2) cDNA into OS3- turned less motile than untransfected cells (Figure 6b R5 cells, and found that GFP-NAG-2 slightly reduced and c, top panel). Remarkably, transfection into the the tumor cell motility, but the difference was not three other SCLC lines, CADO LC6, NCI-H69, and statistically significant (data not shown). OC10, exhibited similar results. When transfected with GFP-CD9, these tumor cells became uniformly less CD9 is absent in SCLC tissues motile than untransfected cells, whereas their motility was not affected when transfected with GFP alone To confirm downregulation of CD9 in SCLC tissues, we (Figure 6c). The inhibition of motility was not complete, stained tumor tissues with anti-CD9 mAb. Representa- but was statistically significant in all the SCLC lines tive tissue specimens were shown in Figure 7. The left

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Figure 4 CD9 and GFP-CD9 associate with CD81 and b1 integrins in OS3-R5 transfectants. (a) Whole cell lysate from OS3-R5 and its transfectants (OS3-R5-pZSV, OS3-R5-pZCD9, OS3-R5-GFP, and OS3-R5-GFP-CD9) were separated on 11% SDS–PAGE and transferred to an Immobilon-P membrane. Western blottingwas performed with anti-CD9 MoAb, MM2/57 (left panel). After stripping, the membrane was reprobed with anti-GFP antibodies (right panel). As a control, lysate of CADO LC9, a NSCLC line, was run in parallel. (b) Immunoprecipitates from lysates of OS3-R5-pZCD9 (left panel) and OS3-R5-GFP-CD9 (right panel) were obtained usinganti-CD9 MoAb, BU16, anti-CD81 MoAb, M38, anti-integrin b1 subunit MoAb, A1A5, anti-N-CAM MoAb, ITK-2, or control IgG. Immunoprecipitated proteins were separated on 11% SDS–PAGE, transferred to a membrane, and Western blotted with biotinylated MM2/57

panel shows typical submucosal invasion of SCLC cells. gression of solid tumors including NSCLC Notably, these invasive SCLC cells did not express CD9. (Higashiyama et al., 1995; Adachi et al., 1996, 1998). Normal epithelium and bronchial glands adjacent to the Since SCLC and NSCLC are proposed to originate from tumors were CD9-positive (Figure 7, left panel). In a common stem cell or an early proliferative cell in the contrast to SCLC, noninvasive, moderately differen- bronchial epithelium. (Mabry et al., 1991), we expected tiated adenocarcinoma cells were clearly CD9-positive that SCLC may also downregulate CD9 or CD82 in (Figure 7, right panel). We stained six more SCLC malignant transformation. The present study, for the specimens, and all these specimens showed negative first time, screened multiple SCLC cell lines for stainingof CD9 (data not shown). tetraspanin expressions. All tetraspanins tested in this study (CD9, CD63, CD81, CD82, CD151, and NAG-2) are present in multiple organs including the lung (Nagira et al., 1994; Fitter et al., 1995; Oritani et al., 1996; Discussion Tachibana et al., 1997; Lantuejoul et al., 1998). The flow cytometry data indicated that, amongthese molecules, Although molecules responsible for the invasion and expressions of CD9 and NAG-2 were less frequent in metastasis of SCLC have been explored, few, if any, SCLC compared with NSCLC. Almost all NSCLC cell have been identified (Shtivelman, 1997; Lantuejoul et al., lines are CD9-positive although the mean fluorescence 1998). In the present study, we explored the possible intensity was variable (Table 1 and data not shown). involvement of tetraspanins in SCLC progression. This observation is consistent with a previous report Accumulatingclinical evidence indicates that down- that examined CD9, CD81, and CD82 expressions in regulation of several tetraspanins correlates with pro- various cancer cell lines, and showed that most NSCLC

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Figure 5 (a) Distribution of GFP or GFP-CD9 after transfection into OS3-R5. OS3-R5 cells placed on FN were transiently transfected with GFP or GFP-CD9 and photographed under an inverted fluorescence microscope. GFP-CD9 was distributed at the cell periphery and at the cell–cell contact (right panel) in contrast to homogenous distribution of GFP (left panel). Magnifications, Â 400 for the larger panels. Enlarged images of transfected cells were shown in the insets. (b) Colocalization of GFP-CD9 and b1 integrins. After the transient transfection, cells were fixed and permeabilized. After treatment of anti-b1 MoAb, localization of GFP- CD9 (left panel), and b1 integrins (middle panel) was analysed using a confocal laser scanning fluorescence microscope. The merged image was shown in the right panel. Colocalization of GFP-CD9 and b1 integrins were observed at the cell periphery (arrowheads) and the cell–cell contact (arrow). Magnification, Â 640 lines (10 of 11) expressed CD9 (White et al., 1998). the significance of difference between SCLC and Amongthe NSCLC lines tested in the present study, NSCLC was smaller than CD9. Ten of 17 SCLC lines only VMRC-LCD lacked CD9. This cell line, however, were not stained with anti-NAG-2 MoAb, but six of 13 might be a variant because N-myc is amplified as seen in NSCLC lines were also clearly negative for NAG-2 SCLC (Kashii et al., 1992). In contrast to NSCLC, CD9 (Table 1). Thus, it seems unlikely that the absence of this was absent or highly reduced in most SCLC lines. The protein accounts for clinical aggressiveness of SCLC. three CD9-positive SCLC lines grew in floating aggre- The precise mechanisms of CD9 downregulation in gates in usual culture condition, but CD9 reactivity did SCLC are not known. However, CD9 gene expression not correlate with cell morphology, because the other was absent or markedly reduced in many SCLC lines, floatingSCLC lines were CD9-negative (Table 1). Also, while there was no difference in another tetraspanin, absence of CD9 does not seem to be a common feature CD63, when compared with NSCLC (Figure 2). Among in cells bearingneuronal characteristics, for this protein SCLC lines tested, OS2-RA had a moderate level of was expressed in normal neuronal tissues (Kaprielian CD9 gene expression despite no reactivity against anti- et al., 1995) and in two neuroblastoma cell lines, IMR- CD9 MoAb (Figure 1 and Table 1). This line may have 32 and NB1 (I Tachibana, unpublished observations). defects in translation or processingof the premature Interestingly, all CD9-positive SCLC lines were also form of CD9. In the other seven SCLC lines, CD9 MHC class I-positive (Table 1). This may reflect reduction probably occurs at or prior to the trans- associated expression between CD9 and class I antigens cription level. Allelic loss of 12p13, at which the CD9 on these cell lines (Lagaudriere-Gesbert et al., 1997). gene is located (Boucheix et al., 1985), was not reported Regarding the absence of another tetraspanin, NAG-2, in SCLC. Also, mutations in the CD9 gene or

Oncogene Tetraspanins in human lung cancer cells T Funakoshi et al 682 hypermethylation in its promoter region have not been (Mashimo et al., 2000). Since tumor suppressor genes demonstrated in cancers. Interestingly, a recent study such as p53 and Rb are more frequently inactivated reported that p53 tumor suppressor protein directly in SCLC than in NSCLC (Salgia and Skarin, activates CD82 gene expression in an NSCLC line, A549 1998), correlation between inactivation of these tumor

Figure 6 GFP-CD9 expression uniformly suppresses cell motility of SCLC lines. (a) Four SCLC lines (open bars) and four NSCLC cell lines (closed bars) were plated onto FN (upper panel)- or PLL (lower panel)-coated wells. The tracks and distances of random motility of at least 30 cells were determined. Each bar represents the mean and standard deviation. (b) OS3-R5 cells were plated onto FN and transiently transfected with GFP (left panel) or GFP-CD9 (right panel). Motility of transfected (fluorescent) and untransfected (nonfluorescent) cells were traced, and tracks of transfected (white line) and untransfected (black line) were visualized usingthe imaging device, MCID, and the program Image Analyzer. The end point of each cell track is indicated by a filled circle. Magnification, Â 40. The results of assay is shown in the top panel of c. (c) Each SCLC line was transiently transfected with GFP (left panel) or GFP- CD9 (right panel). Cell motility of fluorescent ((+), closed bars) and nonfluorescent ((À), open bars) was analysed on FN. Each bar represents the mean and standard deviation

Oncogene Tetraspanins in human lung cancer cells T Funakoshi et al 683 and CD151 (Testa et al., 1999), into a pancreatic cancer cell line and a cervical cancer cell line, respectively, resulted in enhanced cell migration or metastasis. Possibly, the effects on tumor cell motility and metastasis may depend on which tumor cell expresses which tetraspanin. This is compatible with the clinical evidence that CD63 reduction is associated with metastasis of melanoma (Hotta et al., 1988) but not with that of NSCLC (Adachi et al., 1998) or pancreatic cancer (Sho et al., 1998). Here we presented evidence that CD9 functions to inhibit cell motility of an SCLC line, OS3-R5, by two motility assays. Moreover, we performed transient transfection of GFP-CD9 into multiple SCLC lines to exclude the possibility that the reduced motility was a result of clonal selection. Consequently, we observed similar motility-inhibitory effects of this molecule in all SCLC lines, although the inhibition was not complete. These results suggest that, at least partially, decreased expression of CD9 may be responsible for the highly invasive property of SCLC. We assumed that GFP-CD9 would function in the same way as untagged CD9 for the following reasons. First, GFP-CD9 as well as untagged CD9 associated with b1 integrins and CD81 (Figure 4b). Although we used mild detergent Brij 99 to show this association, absence of CD9 or GFP-CD9 in control or anti-N- CAM immunoprecipitation, at least assures that this association is not nonspeciﬁc. Second, GFP-CD9 was distributed and colocalized with b1 integrins at the cell periphery and cell–cell contacts (Figure 5). This distribution of GFP-CD9 was consistent with previously reported intracellular distribution of CD9 (Nakamura et al., 1995; Berditchevski and Odintsova, 1999). Third, the stable transfectant, OS3-R5-GFP-CD9, was as static as OS3-R5-pZCD9 when compared with the parent OS3-R5 or OS3-R5-GFP on FN-coated Transwells (data not shown). Moreover, the time-lapse videomicroscopic cell motility assays also revealed that GFP-CD9 inhibits cell motility (Figure 6c) as well as untagged CD9 in OS3-R5 (Figure 3d). A videomicroscopic assay after transfection of a ﬂuorescein-tagged cDNA similar to the present one might be useful to screen various tumor lines to see if a given tetraspanin protein is motility-inhibitory or not. It is well accepted that tetraspanin forms complexes with integrins and modulates integrin functions to Figure 6 Continued regulate cell motility (Hemler et al., 1996; Hemler, 1998). SCLC is surrounded by an extensive stroma of extracellular matrix includingFN at both primary and suppressors and downregulation of tetraspanins in lung metastatic sites (Sethi et al., 1999). Most lungcancer cell cancer should be explored. In this respect, it is of note lines as well as normal bronchial epithelial cells express that reconstitution of the Rb gene into a Rb-defective b1 integrins, and among known a and b integrins, a3b1 NSCLC cell inhibited tumor cell invasion in vitro (Li was uniformly expressed in SCLC lines in previous et al., 1996). studies (Feldman et al., 1991; Sethi et al., 1999). Although a previous study showed that overexpres- Although a3b1 does not mediate strongadhesion to sion of CD9 into an NSCLC line suppressed tumor cell FN, a3b1-dependent cell migration on FN has been motility (Ikeyama et al., 1993), another study presented previously reported (Yauch et al., 1998). SCLC also contradictory evidence that CD9 transfection into a B- expresses another adhesion molecule, N-CAM, which cell line upregulated cell motility on FN and LM (Shaw mediates homophilic cell–cell adhesion, and may be et al., 1995). In addition, other studies reported that involved in SCLC metastasis (Scheidegger et al., 1994). transfection of tetraspanins, CO-029 (Claas et al., 1998) In the present study, however, the exogenous CD9 and

Oncogene Tetraspanins in human lung cancer cells T Funakoshi et al 684

Figure 7 Immunohistochemical stainingof CD9 in lungcancer tissues. Primary tumor tissues of SCLC (left panel) and lung adenocarcinoma (right panel) were stained with a monoclonal antibody to CD9. The left panel shows typical submucosal invasion of SCLC cells. These SCLC cells were CD9-negative, while normal epithelium and bronchial glands adjacent to the tumor were CD9- positive. Moderately differentiated adenocarcinoma cells in the right panel were CD9-positive. Magniﬁcation, Â 50

GFP-CD9 was associated with b1 integrins, but not with In summary, we have shown that, amongtetraspa- N-CAM in the OS3-R5 transfectants. Thus, when nins, CD9 expression is selectively downregulated in introduced into SCLC cells, CD9 and GFP-CD9 SCLC but not in NSCLC, and that recovery of CD9 presumably complex with and regulate b1 integrins to expression suppresses cell motility of SCLC. The reduce cell motility on FN. difference in clinical aggressiveness between SCLC and In contrast to NSCLC, SCLC easily spreads into NSCLC may be, at least in part, because of the absence submucosal tissues and metastasizes to regional lymph of this motility-related protein in SCLC. nodes. Our in vitro data have raised the hypothesis that downregulation of CD9 may play a role in the early invasion of SCLC. We further considered in vivo Materials and methods spontaneous metastasis experiments usingstable transfectants in nude mice. However, none of these SCLC Cell lines lines formed metastatic foci when inoculated subcuta- NCI-H69, NCI-N231, NCI-N857, Lu65, and Lu99, were gifts neously. The immunohistochemistry data may instead from Dr Y Shimosato, National Cancer Research Institute, support our hypothesis. While CD9 is expressed in Tokyo, Japan. SBC-1, RERF-LC-MS, PC-3, VMRC-LCD, adenocarcinoma of the lung, all seven primary SCLC and EBC-1 were obtained from the Japanese Collection of tumors tested were CD9-negative (Figure 7 and data not Research Bioresources (Tokyo, Japan). OC10, CADO LC6, shown). It was reported that CD9 and CD82 were and CADO LC9 were provided by Osaka Medical Center for variably expressed in resected primary tumors of Cancer and Cardiovascular Diseases, Osaka, Japan. Seven NSCLC, and that low expression of these tetraspanins SCLC cell lines, OS1, OS2-RA, OS3-R5, OS4, OS5, OS6, and correlated with poor prognosis of the patients (Higa- OS7, were established in our laboratory, and their biological properties were previously characterized (Tanio et al., 1992; shiyama et al., 1995; Adachi et al., 1996, 1998). Saito et al., 1994). Four additional SCLC lines, OS10, OS15, Moreover, CD9 reduction was more frequently ob- OS17, and OS18A, a lungadenocarcinoma line, OAD2, and a served in metastatic lymph nodes than primary tumors lungsquamous cell carcinoma line, OSQ1, were also estab- in breast cancers (Miyake et al., 1995). Thus, it is lished in our laboratory. A549, QG56, and neuroblastoma probable that a portion of cells loses CD9 expression lines, IMR-32 and NB1, were obtained from the American and acquires advantages for invasion and metastasis Type Culture Collection, Rockville, MD, USA. PC9 was in the late stage of progression in NSCLC, whereas donated by Dr Y Hayata, Tokyo Medical College, Tokyo, downregulation of CD9 occurs in the much earlier Japan. HARA was a kind gift from Dr H Iguchi, Kyusyu stage of malignant transformation of SCLC. These Cancer Center, Fukuoka, Japan. All cell lines were maintained two types of lungcancers may have distinct mecha- in RPMI 1640 medium supplemented with 10% heat- inactivated fetal calf serum (FCS), 100 U/ml penicillin, and nisms of CD9 downregulation, which lead to distinct 100 mg/ml streptomycin, and were free of Mycoplasma infec- clinical behaviors. Since SCLC initially responds tion when tested with Bacto pleuropneumonia-like organism well to chemoradiotherapy but later most of the tumors agar (Difco Laboratories, Detroit, MI, USA). All SCLC or recur around the primary sites and metastasize to NSCLC lines described above were examined for N-CAM distant organs, suppression of the early invasion is expression by ﬂow cytometry prior to experiments, and important to overcome the spread of SCLC. In a recent conﬁrmed to be N-CAM-positive or -negative, respectively study, adenovirus-mediated CD9 delivery into an (data not shown). inoculated murine melanoma markedly inhibited pulmonary metastasis and prolonged survival of the Antibodies mice (Miyake et al., 2000). Thus, CD9 may be a Mouse anti-CD9 MoAbs, BU16 and MM2/57, were purchased promising candidate for a novel gene-delivery strategy from Biodesign International (Kennebunk, ME, USA) and against SCLC. Biosource International (Camarillo, CA, USA), respectively.

Oncogene Tetraspanins in human lung cancer cells T Funakoshi et al 685 M38 and M104, mouse MoAbs against CD81 and CD82, lines were transiently transfected with GFP-tagged CD9 or respectively, were gifts from Dr O Yoshie (Kinki University GFP alone, and analysed in a time-lapse videomicroscopic cell School of Medicine, Osaka, Japan). 6H1, 5C11, and NAG-1, motility assay. mouse MoAbs against CD63, CD151, and NAG-2, respectively, and A1A5 and SG/19, mouse anti-b1 integrin MoAbs, Western blotting were previously described (Tachibana et al., 1996; Berditch- evski et al., 1997; Tachibana et al., 1997; Yauch et al., 1998). A Cells were lysed at 41C for 1 h in analysis buffer containing1% m m m mouse anti-N-CAM MoAb, ITK-2, was established in our Brij 99, 25 m HEPES, pH 7.5, 150 m NaCl, 5 m MgCl2, laboratory and characterized previously (Saito et al., 1991). A 2mm phenylmethylsulfonyl fluoride,10 mg/ml aprotinin, and mouse anti-MHC class I MoAb, W6/32, was provided by Dr 10 mg/ml leupeptin. Samples containing an equal protein ME Hemler (Dana-Farber Cancer Institute, Boston, MA, concentration were mixed with an equivalent volume of USA). Another mouse anti-MHC class I MoAb, IOT2, was sample buffer (50 mm Tris/HCl, pH 6.5, 10% glycerol, 2% purchased from Immunotech, ME, USA. Rabbit anti-GFP SDS, and 0.1% bromophenol blue), boiled for 5 min, antibodies and normal mouse IgG were purchased from electrophoresed on 11% SDS–polyacrylamide gels under Clontech (Palo Alto, CA, USA) and Serotec (Kidlington, nonreducingconditions, and transferred to Immobilon-P England), respectively. membranes (Millipore, Bedford, MA, USA). After washes, nonspecific bindingsites of the membranes were blocked in PBS containing30 mg/mlnonfat skimmed milk for 30 min. Flow cytometry Then the membranes were probed with an anti-CD9 MoAb, Ten thousand cells were stained with ITK-2 (anti-N-CAM), MM2/57, diluted at 1 mg/ml by shaking overnight at 41C, W6/32 (anti-MHC class I), MM2/57 and BU16 (anti-CD9), followed by 1-h incubation with peroxidase-conjugated goat 6H1 (anti-CD63), M38 (anti-CD81), M104 (anti-CD82), 5C11 anti-mouse IgG (H+L) (BIO-RAD, Hercules, CA, USA) (anti-CD151), or NAG-1 (anti-NAG-2) at a concentration of diluted 1 : 2000. Immunoreactive bands were visualized by 10 mg/ml, washed twice, and then labeled with fluorescein treatingwith Renaissance Chemiluminescent Reagent(NEN TM isothiocyanate (FITC)-conjugated goat anti-mouse immuno- Life Science Products, Boston, MA, USA). After MM2/57 was globulin (Biosource International). Normal mouse IgG was stripped, the membrane was reprobed with rabbit anti-GFP used as a control. Stained cells were analysed on a FACSort antibody (Clontech). (Becton-Dickinson, San Jose, CA, USA). Immunoprecipitation RT–PCR analysis Whole cell lysates were precleared by incubation with protein A-Sepharose beads (Sigma, St Louis, MO, USA) for 1 h at Total RNA was extracted from cultured lungcancer cell lines 41C. Then the lysates containingequal amounts of protein with Isogen (Nippon Gene, Tokyo, Japan). A volume of 1 mg were rotated for 1 h at 41C with mouse anti-CD9 MoAb, of total RNA was reverse transcribed usingOne Step RNA BU16, anti-CD81 MoAb, M38, anti-b1 integrin MoAb, A1A5, PCR Kit (Takara, Kyoto, Japan) followingthe manufacturer’s anti-N-CAM MoAb, ITK-2, and normal mouse IgG. Immune instructions. The generated cDNAs were amplified using complexes were collected by an additional rotation with primers for CD9 (50-TGCATCTGTATCCAGCGCCA-30 protein A-Sepharose beads for 1 h at 41C. After washingthree and 50-CTCAGGGATGTAAGCTGACT-30) (Higashiyama times with lysis buffer and boilingfor 5 min in sample buffer, et al., 1995; Miyake et al., 1995) and CD63 (50- immunoprecipitated proteins were separated on 11% SDS– CCCGAAAAACAACCACACTGC-30 and 50-GATGAG- polyacrylamide gel electrophoresis (PAGE) under nonreducing GAGGCTGAGGAGACC-30) (Adachi et al., 1998). b-actin conditions and transferred to an Immobilon-P membrane. cDNA amplification was used as the internal control. Primers Western blottingwas performed by incubation with biotiny- used for b-actin were 50-CAAGAGATGGCCACGGCTGCT- lated anti-CD9 MoAb, MM2/57, followed by peroxidase- 30 and 50-TCCTTCTGCATCCTGTCGGCA-30. These primer conjugated streptavidin (Zymed, San Francisco, CA, USA). pairs for CD9, CD63, and b-actin amplify 799-, 347-, and 274- Bands were visualized with Renaissance Chemiluminescent bp fragments, respectively. The reaction mixtures were Reagent as described above. subjected to 25 PCR amplification cycles each consistingof 40 s at 941C, 40 s at 601C, and 90 s at 721C. The amplified DNA samples were electrophoresed on 1% agarose gels and Cell adhesion assay visualized with ethidium bromide. We confirmed that para- Human plasma FN (GibcoBRL), murine EHS sarcoma meters on these RT–PCR conditions yielded amplification of laminin-1 (LM) (Sigma), and BSA (Sigma) were diluted in template DNAs within a linear range. 0.1 m sodium bicarbonate at a concentration of 20 mg/ml. Wells in a 96-well nontissue-culture-treated plate (Linbro, McLean, cDNA transfection VA, USA) were precoated with 100 ml of the solution overnight at 41C. Nonspecific bindingsites were then blocked with 100 ml The coding region of CD9 cDNA was generated by RT–PCR of PBS, 0.1% BSA at 371C for 2 h. After washingthree times and cloned in frame into expression vectors, pZeoSV (Invitro- with PBS, 1.5 Â 104 cells resuspended in 100 ml of RPMI 1640 gen, Carlsbad, CA, USA) and pEGFP-C1 (Clontech). In the were allowed to adhere to the FN-, LM-, or BSA-coated wells latter, CD9 cDNA was fused to the carboxyl terminus of GFP for 1.5 h at 371C. Unattached cells were eliminated by shaking, with the addition of a nine-amino-acid polylinker (SGLRSRA- and then the remainingadherent cells were evaluated usinga 3- QA). OS3-R5-pZCD9 and OS3-R5-GFP-CD9 were estab- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide lished by transfection of these constructed vectors into OS3-R5 (MTT) assay as previously described (Tachibana et al., 1996). usingLipofectAMINE 2000 Reagent(GibcoBRL, Rockville, MD, USA) followed by selection in Zeocin (Invitrogen) and Boyden chamber cell motility assay G-418 sulfate (GibcoBRL), respectively. OS3-R5-pZSV and OS3-R5-GFP were similarly established by transfection with Tissue-culture-treated Transwell membranes (pore size, 8 mm, the vectors alone. In other experiments, several other SCLC Costar, Cambridge, MA, USA) were precoated with FN or

Oncogene Tetraspanins in human lung cancer cells T Funakoshi et al 686 PLL as described above. Cells (5 Â 104) resuspended in 100 ml goat anti-mouse IgG. Localization of GFP-CD9 and b1 of RPMI 1640, 10% FCS were applied to the upper chamber integrins were analysed using a confocal laser scanning of Transwells. A volume of 600 ml of RPMI 1640, 10% FCS fluorescence microscope (Carl Zeiss, Thornwood, NY, USA). were added to the lower chamber. After incubation for 18 h at 371C, cells migrating through the membrane to the lower Immunohistochemistry chamber and adheringto the bottom were counted. For antibody blockingexperiments, OS3-R5 cells were preincu- The immunoperoxidase procedures (ABC method) usinga bated with 10 mg/ml of antibodies for 30 min, and then directly monoclonal antibody to CD9 (Novocastra, Newcastle, UK; applied to the upper chamber of Transwells. diluted 1 : 20) was carried out in transbronchial biopsy specimens of seven cases of SCLC and one case of lung adenocarcinoma as previously described (Hsu et al., 1981). For Time-lapse videomicroscopic cell motility assay antigen retrieval, sections were pretreated with microwaving One thousand cells suspended in 100 ml of RPMI 1640, 10% for 15 min in 1 mm EDTA (pH 8.0) before incubation. FCS were plated onto an FN-, or PLL-precoated wells in a 96- well plate and cultured for 24 h before monitoring. The cells Statistical analysis were then incubated at 371C, 10% CO2, and monitored using an inverted fluorescence microscope, Diaphoto (Nikon, Statistical significance of differences in the number of positive Tokyo, Japan) equipped with an SVHS video recorder (Sony, lines for each tetraspanin expression between SCLC and Tokyo, Japan) for 2 h (Kashii et al., 1992; White et al., 1998). NSCLC was determined by w2 test. The tracks and distances of random motility of at least 30 cells were obtained by the microcomputer imaging device (MCID) (Imaging Research, Ontario, Canada) using the program, Acknowledgments Image Analyzer (Ver. 3) (Imaging Research). This work was partially supported by a grant from Sankyo Foundation of Life Science, Tokyo, Japan, and by Grants-in- Aid for Cancer Research from the Ministry of Education, Immunofluorescence Science and Culture, Japan. The authors are grateful to Dr OS3-R5 cells cultured on FN were transiently transfected with Martin E Hemler (Dana-Farber Cancer Institute, Boston) and GFP-tagged CD9 as described above, and incubated for 24 h. Dr Osamu Yoshie (Kinki University School of Medicine, After washes, cells were fixed with PBS, 4% paraformaldehyde Osaka, Japan), for providingantibodies to tetraspanins. We for 15 min, permeabilized with PBS, 0.1% Triton X-100, and also thank Yasuo Katsuki and Eiji Oiki (Osaka University blocked with PBS, 3% BSA for 30 min at room temperature. Graduate School of Medicine) for technical assistance on the Immunofluorescent stainingwas conducted with anti- b1 time-lapse videomicroscopic cell motility assay and Yoko integrin MoAb, SG/19, followed by rhodamine-conjugated Habe for secretarial assistance.

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