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

Oncogene (2003) 22, 674–687 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc 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

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