British Journal of Cancer (2003) 89, 158 – 167 & 2003 Cancer Research UK All rights reserved 0007 – 0920/03 $25.00 www.bjcancer.com Clinical significance of transmembrane 4 superfamily in colon cancer 1,5 2 1 1 1 3 5 4 H Hashida , A Takabayashi , T Tokuhara , N Hattori , T Taki , H Hasegawa , S Satoh , N Kobayashi , 5 ,1 Y Yamaoka and M Miyake* 1 Department V of Oncology and Department of Thoracic Surgery, Kitano Hospital, Tazuke Kofukai Medical Research Institute, 2-4-20, Ohgimachi, 2 Kita-ku, Osaka 530-8480, Japan; Department of Surgery, Kitano Hospital, Tazuke Kofukai Medical Research Institute, 2-4-20, Ohgimachi, Kita-ku, Osaka, 530-8480, Japan; 3First Department of Internal Medicine, Ehime University School of Medicine, Oazashizukawa, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan; 4First Department of Surgery, Ehime University School of Medicine, Oazashizukawa, Shigenobu-cho, Onsen-gun, Ehime 791- 5 0295, Japan; Department of Gastroenterological Surgery, Kyoto University Graduate School of Medicine, 54, Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan Cell motility is an important cellular function closely related to the processes of tumour progression and metastasis. Several members of transmembrane 4 superfamily (TM4SF) have been reported to be associated with cell motility and metastatic potential of solid tumour. The aim of this study is to clarify the clinical significance of the member of TM4SF (MRP-1/CD9, KAI1/CD82 and CD151) in Molecular and Cellular Pathology human colon cancer. We studied 146 colon cancer patients who underwent curative surgery and studied the expression of MRP-1/ CD9, KAI1/CD82 and CD151 using reverse transcriptase – polymerase chain reaction and immunohistochemistry. We found that 64 patients (43.8%) had MRP-1/CD9-positive tumours and that the overall survival rate of patients with MRP-1/CD9-positive tumours was much higher than that of patients with MRP-1/CD9-negative tumours (89.8 vs 50.8%, Po0.001). In contrast, 63 patients (43.2%) had KAI1/CD82-positive tumours and the overall survival rate of patients with KAI1/CD82-positive tumours was also higher than that of patients with KAI1/CD82-negative tumours (84.8 vs 54.9%, P ¼ 0.002). On the other hand, positive CD151 expression had a bad effect on the overall survival rate of patients with colon cancer (61.2 vs 74.9%, P ¼ 0.022). In a multivariate analysis, MRP-1/CD9 status was a good indicator of the overall survival (P ¼ 0.007). We have shown that the reduction of MRP-1/CD9 and KAI1/CD82 expression, and the increasing CD151 expression are indicators for a poor prognosis in patients with colon cancer. This is a first report describing about the relation between CD151 and colon cancer. British Journal of Cancer (2003) 89, 158–167. doi:10.1038/sj.bjc.6601015 www.bjcancer.com & 2003 Cancer Research UK Keywords: TM4SF; MRP-1/CD9; KAI1/CD82; CD151; colon cancer Cell motility plays an important key function related to the process of unequal size, as well as a particular fold in the large extracellular of tumour progression and metastasis (Miyake and Hakomori, loop (Wright and Tomlinson, 1994). 1991). It is partially dependent on adhesion molecules and KAI1/CD82 is also a member of TM4SF. KAI1/CD82 expression proteases (Hashida et al, 2001, 2002a). Previously, we reported suppressed experimental metastasis of rat prostate tumour cells that the motility-related protein-1 (MRP-1) is an antigen (Dong et al, 1995), and decreased motility and invasion of colon recognised by monoclonal antibody (MAb) M31-15 which inhibits carcinoma cells (Takaoka et al, 1998). KAI1/CD82 is considered to cell motility and the MRP-1 sequence coincides with the cluster of be a metastasis-suppressor gene of prostate cancer and low KAI1/ differentiation antigen 9 (CD9) (Miyake et al, 1991). In our CD82 expression has been reported to be involved in the malignant previous reports, we showed MRP-1/CD9-overexpressing tumour progression of prostate cancer (Dong et al, 1996). We also showed cells negative cell motility and metastatic potential (Ikeyama et al, that decreased KAI1/CD82 gene expression was an indicator of 1993). Therefore, MRP-1/CD9 regulates cell motility and is a poor prognosis in lung cancer (Adachi et al, 1996), breast cancer receptor for negative signal ligands. In addition, negative MRP-1/ (Huang et al, 1998) and pancreatic cancer (Sho et al, 1998). These CD9 expression was associated with a poor prognosis in breast data show that KAI1/CD82 is an important tumour suppressor cancer (Miyake et al, 1996), lung cancer (Higashiyama et al, 1995) gene in cancer metastasis and progression. and pancreatic cancer (Sho et al, 1998). These data also suggest On the other hand, CD151 is also a transmembrane molecule that MRP-1/CD9 expression might be associated with metastatic that has been characterised as a member of the evolutionally ability and degree of malignancy. MRP-1/CD9 belongs to the conserved TM4SF, and it is known as SFA-1 and PETA-3 (Fitter transmembrane 4 superfamily (TM4SF), which is characterised by et al, 1995; Hasegawa et al, 1996). CD151 cDNA shows an open four transmembrane domains delimiting two extracellular regions reading frame of 253 amino acids that encodes a protein of molecular mass 28 kDa. In addition, human CD151 gene locates on *Correspondence: Dr M Miyake; E-mail: [email protected]. chromosome 11p15.1. CD151 in involved in cell adhesion, cell Received 18 October 2002; revised 12 March 2003; accepted 23 March motility, metastasis, and stability and formation of hemidesmo- 2003 somes (Yauch et al, 1998). TM4SF in colon cancer H Hashida et al 159 Several members of TM4SF are associated with the metastatic patients were men and 62 were women. The median age of the phenotype. In addition, for the most part, they work negatively. patients was 62.8 years, with a range of 35–80 years. The patients Recently, it was reported to be the first member of the TM4SF to could be broken down into 25 with pathological stage I, 46 with show signs of being a positive effecter of metastasis (Testa et al, stage II and 75 with stage III disease. The mean follow-up period 1999). Moreover, CD151 enhances cell motility and cancer for all patients was 44.3 months, with a range of 6.3–85.9 months. metastasis (Kohno et al, 2002) and CD151 overexpression leads to a poor prognosis of the patients with non-small cell lung cancer (Tokuhara et al, 2001). Therefore, CD151 is a metastasis-associated Tumour specimens antigen that appears to contribute to the metastatic phenotype One-half of each fresh tumour tissue specimen was immediately positively. These findings set CD151 apart from MRP-1/CD9 and embedded in optimum cutting temperature compound (Miles, KAI1/CD82 that appear to act as metastasis-suppressor genes. It Kankakee, IL, USA), and frozen in liquid nitrogen immediately may contribute to the collapse of tetraspanin/tetraspanin com- after surgical resection and maintained at À801C until use. Frozen plexes. In addition, no consistent findings have been reported as a sections were cut on a cryostat to a thickness of 6 mm and were prognostic indicator for CD151 gene in colon cancer. As part of stained with haematoxylin and eosin and used for immunohisto- our evaluation of members of the TM4SF as possible prognostic chemical staining. After the connective tissues were trimmed off, predictors, we performed a retrospective study on the expression the other-half of the tumour specimen that was then made up of of the MRP-1/CD9 gene, the recently identified KAI1/CD82 gene more than 80% cancer cells was used for the reverse transcriptase– and the CD151 gene in human colon cancer. polymerase chain reaction (RT–PCR) analysis. MATERIALS AND METHODS Immunohistochemical assays Clinical characteristics of the patients The assays were carried out as described previously (Hashida et al, 2002a). Endogenous peroxidases were blocked by incubating with We studied 146 patients with up to stage III colon cancer who had 0.3% H2O2 in absolute methanol for 30 min. The sections were then undergone surgery at the Department of Surgery of the Kitano incubated with 5% bovine serum albumin for 2 h at room Hospital between October 1994 and May 2001. All patients temperature. Subsequently, replicate sections were incubated for underwent curative surgery. The postsurgical staging of each 2 h with the anti-MRP-1/CD9 MAb M31-15, the anti-KAI1/CD82 tumour was classified according to the tumour–node–metastasis MAb C33 and the anti-CD151 MAb SFA1.2B4, respectively. After (TNM) staging system (Sobin and Wittekind, 1997). The clinical washing three times in phosphate-buffered saline (PBS), they were characteristics of the patients are presented in Table 1. In all, 84 of then incubated for 1 h with biotinylated horse anti-mouse IgG Table 1 Relation of MRP-1/CD9, KAI1/CD82 and CD151 expression and various prognostic factors in 146 patients with colon cancer MRP-1/CD9 KAII/CD82 CD151 Clinicopathological Number of characteristics patients Positive Negative P-value Positive Negative P-value Positive Negative P-value Age (years) p60 74 35 39 NS 33 41 NS 35 39 NS X60 72 29 43 30 42 46 26 Sex Female 62 29 33 NS 29 33 NS 32 30 NS Male 84 35 49 34 50 49 35 Molecular and Cellular Pathology Tumour status T1 17 9 8 NS 11 6 NS 9 8 0.025 T2 20 8 12 7 13 12 8 T3 83 38 45 38 45 39 44 T4 26 9 17 7 19 21 5 Nodal status N0 71 39 32 0.007 43 28 o0.001 37 34 NS N1 33 14 19 11 22 15 18 N2 42 11 31 9 33 29 13 Pathological stage I 25 13 12 0.030 15 10 o0.001 15 10 NS II 46 26 20 28 18 22 24 III 75 25 50 20 55 44 31 Differentiation Well 32 16 16 NS 18 14 NS 14 18 NS Moderately 101 42 59 40 61 57 44 Poorly 13 6 7 5 8 10 3 Total 146 64 82 63 83 81 65 NS ¼ not significant.