World J Gastroenterol. 2004 August 1; 10(15): 2245-2249.
Published online 2004 August 1. doi: 10.3748/wjg.v10.i15.2245.
KAI1 gene expression in colonic carcinoma and its clinical significances
De-Hua Wu, Li Liu, Long-Hua Chen and Yan-Qing Ding.
De-Hua Wu, Long-Hua Chen, Department of Radiation Oncology, Nanfang Hospital, First Military Medical University, Guangzhou 510515, Guangdong Province, China
Li Liu, Yan-Qing Ding, Department of Pathology, First Military Medical University, Guangzhou 510515, Guangdong Province, China
Author contributions: All authors contributed equally to the work.
Correspondence to: Dr. Yang-Qing Ding, Department of Pathology, First Military Medical University, Guangzhou 510515, Guangdong Province, China.
Telephone: +86-20-61642148 Fax: +86-20-61642148
Received December 12, 2003; Revised December 23, 2003; Accepted February 1, 2004;
AIM: To investigate KAI1 gene expression in the progression of human colonic carcinoma and its clinical significances.
METHODS: KAI1 expression was detected by in situ hybridization and immunohistochemistry in the 4 established cell lines of colorectal carcinoma with different metastatic potentials, and in 80 specimens of colonic carcinoma, 21 colonic carcinoma specimens with lymphatic metastasis and 20 controls of normal colonic mucosa.
RESULTS: The expressions of KAI1 in HT29 and SW480 cell lines were higher than those in LoVo and SW620. The expression of KAI1 gene was significantly higher in colorectal carcinoma compared with normal colonic mucosa and lymphatic metastasis (χ2 = 46.838, P < 0.01). The expression of KAI1 gene had no relationship with histological grade. The KAI1 expressions in Dukes A and B carcinoma were higher at both mRNA and protein levels compared to Dukes C carcinoma (χ2 = 16.061, P < 0.05). The expression of KAI1 in colonic carcinoma specimens with lymphatic metastasis was almost lost. The results of in situ hybridization were in concordance with immunohistochemistry.
CONCLUSION: KAI1 is highly related to the metastasis of colonic carcinoma and may be a useful indicator of metastasis in colonic carcinoma.
Colorectal carcinoma is one of the most common forms of malignancy, and metastasis is the major cause of mortality in human population. Timely and precise identification of the occurrence of metastasis and its contributive factors are very important in the enhancement of prognosis and effects of treatment. KAI1 gene was first identified in human prostate carcinoma and mapped to human chromosome 11p11.2[1]. It encodes 267 amino acids belonging to plasma membrane glycoprotein, which consists of 4 transmembrane domains and 1 large and 1 small extracellular domains. The extracellular domains have 3 potential N-glycosylation sites. According to its special structure, it may be predicted that the function of KAI1 gene may come down to cell-cell adherence and cell-matrix connection[2]. The role of KAI1 in tumor progression may not be limited to prostatic cancer. It was reported to be important in preventing the development of metastases in a wide variety of human tumor types, including cervical[3], breast[4], pancreatic[5], esophageal[6], bladder [7], and ovarian cancers[8]. In the present study, the expression of KAI1 was detected by in situ hybridization in 4 cell lines of colorectal carcinoma, and in paraffin-embedded normal colonic epithelium, carcinoma, and lymphatic metastasis. The purpose was to explore the relationship between KAI1 expression and colonic carcinoma metastasis and its clinical significances.
Cell lines and culture conditions
LoVo and HT29 cell lines were cultured in RPMI 1640 supplemented with 100 mL/L heat-inactivated fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin. SW480 and SW620 were cultured in DMEM medium supplemented with the 100 mL/L FBS. All cells were grown in 50 mL/L CO2 humidfied atmosphere at 37 °C.
Tissue specimens
A total of 80 colonic carcinoma specimens, 20 normal colonic mucosa samples and 20 lymphatic metastasis samples were obtained from Nanfang Hospital. All the samples were routinely fixed in 40 g/L formaldehyde solution, embedded in paraffin, and cut into 4 m thick sections. Samples were selected according to the pathological diagnosis and reviewed by a pathologist to confirm the diagnosis.
In situ hybridization (ISH) assays
The digoxigenin -labeled KAI1-cDNA probe for in situ hybridization was prepared as described in one of our previous papers[9]. The ISH was performed according to the instruction of the enhanced sensitive ISH detection kit (purchased from Boster Biotechnology Company). Briefly, the sections were deparaffinized with xylene, dehydrated with graded ethanol and incubated in 30 mL/L H2O2 to block endogenous peroxidases for 30 min at room temperature. After being treated for 10 min with Triton X-100 and for 40 s with 3% pepsin, the sections were prehybridized for 3 h and hybridized overnight at 37 °C. The final concentrations of the labeled probes were 0.15 ng/L. After hybridization, excess probes were removed through rinsing in 2 × SSC (1 × SSC is 0.15 mol/L NaCl plus 0.015 mol/L sodium citrate pH 7.0), 0.5 × SSC, 0.2 × SSC, respectively. The sections were incubated with an antidigoxigenin antibody conjugated biotin for 120 min at room temperature and then added strept-avidin biotin complex for 30 min. For color reaction, diaminobenzidine (DAB) was used. If the ISH signals were present, the cytoplasm would be full of brown granules. For ISH of the four colorectal carcinoma cell lines, the cells were incubated and grown on cover slip for 24 h, fixed with 950 mL/L ethanol and washed with PBS (phosphate-buffered saline pH 7.2) 3 times, and then carried out ISH as described above. For negative controls, the digoxigenin-labeled KAI1-cDNA probe was replaced by prehybridized solution.
Immunohistochemical assays
To confirm the results of KAI1 gene expression on in situ hybridization, immunohistochemical studies were performed as described previously[9,10]. Briefly, the fixed cells and sections were subjected to immunostaining by using an ultrasensitive streptavidin -peroxidase technique (Maixin Biotechnology Company). Endogenous peroxidases were blocked by incubating with 5 mL/L H2O2 for 30 min at room temperature. The cells and sections were subsequently treated for 10 min with Triton X-100 and for 40 s with 30 g/L pepsin. The cells and sections were incubated for 30 min at 37 °C with normal nonimmune serum before overnight incubation at 4 °C with specific monoclonal KAI1-antibody (BD pharmingen technical company) at a dilution of 1:100. The cells and sections were then treated with biotin-conjugated second antibody before adding streptavidin-peroxidase. For color reaction, diaminobenzidine was used. If the positive signals were present, the cytoplasm and membrane were stained brown. For negative controls, the monoclonal KAI1 antibody was replaced by PBS
Review and scoring of the section
The stained sections were reviewed and scored indepentdently by two pathologists under Olympus microscope. The different degrees of intensity of staining were graded on a scale of 0 to 3 as follows: 0 indicated that the number of positive cells was less than 10%; 1 indicated weak positive and the number of positive cells more than 10%, but less than 30%; 2 indicated positive and the number of positive cells more than 30% but less than 50%; 3 indicated strong positive and the number of positive cells more than 50%.
Statistical analysis
Fisher’s exact probability test was adopted to examine the relationship between the variables. A P value < 0.05 was considered statistically significant.
KAI1 gene expression in colorectal carcinoma cell lines
HT29 and SW480 cell lines were derived from primary colorectal adenocarcinoma. SW620 and LoVo cell lines were established from metastatic colorectal carcinoma. Especially, SW620 cell line was isolated from the same case as was SW480 cell line. The expressions of KAI1 mRNA and protein in HT29 and SW480 cells were positive, whereas those in SW620 and LoVo cells were negative (Figure 1, Figure 2).
Figure 1
Figure 1
Figure 1
In situ hybridization detection of expression of KAI1 mRNA in colorectal carcinoma cell lines (Original magnification: × 400). A: In HT29 cells, the positive expression (brown granule) was located in cytoplasm; B: In SW480 cells, the positive expression was located in cytoplasm; C: In SW620 cells, the expression of KAI1 mRNA was negative; D: In LoVo cells, the expression of KAI1 mRNA was negative.
Figure 2
Figure 2
Figure 2
Expression of KAI1 protein detected by immunohistochemistry in colorectal carcinoma cell lines (Original magnification: × 400). A: In HT29 cells, the positive expression located in cytoplasm and membrane; B: In SW480 cells, the positive expression located in cytoplasm and membrane.
Heterogeneous expression of KAI1 mRNA in colonic carcinoma tissue
Of the 80 colonic carcinoma specimens, 56(70%) were classified as KAI1 positive (Figure 3). There were 4 cases (20%) with positive KAI1 mRNA expression in normal colonic mucosa and 2 cases (10%) in lymphatic metastasis. The results are shown in Table 1. The positivity ratio was significantly higher in colonic carcinoma than that in nomal colonic mucosa and in lymphatic metastasis (χ2 = 46.838, P < 0.01). The expression in lymphatic metastasis was almost lost.
Table 1
Table 1
KAI1 gene expression in normal colonic mucosa, colonic carcinoma and lymphatic metastasis n (%)
Figure 3
Figure 3
Figure 3
In situ hybridization and immunohistochemical detection of KAI1 mRNA and protein in colonic carcinoma (Original magnification: × 400). A: In situ hybridization detection found that strong KAI1 mRNA staining was located in cytoplasm (brown granule); B: Detection by immunohistochemistry showed strong KAI1 protein staining was in cytoplasm and membrane.
To further investigate the relationship between the expression of KAI1 and the clinical pathology, we sorted the colonic carcinomas based on histological grades and Dukes stages. Histological grade I means well differentiated adenocarcinoma, II means moderatedly differentiated and III means poorly differentiated. The results indicated that KAI1 expression had no relationship with histological grades (χ2 = 3.887, P > 0.05). The expression in Dukes A and B carcinoma was markedly higher in comparison with Dukes C carcinoma (χ2 = 16.061, P < 0.01).
KAI1 protein expression analyzed by immunohistochemistry
There were 52(65%) cases with positive KAI1 protein expressions in the colonic carcinomas (Figure 3). The positive ratios of nomal colonic mucosa and lymphatic metastasis were 25% and 10%, respectively. The KAI1 expressions in 3 groups were significantly different (χ2 = 28.298, P < 0.01). Overall, the immunohistochemical results agreed well with those from the in situ hybridization assays.
Loss of the function of metastasis suppressor genes is an important step in the progression of a tumor type. Several candidate antimetastasis or anti -invasion genes have been studied in colorectal carcinoma, including nm23[11], E-cadherin[12], and CD44[13], but inconsistent findings have been reported. For example, in separate studies, nm23 expression has been found to be directly correlated, not to be correlated, or inversely correlated with metastatic potential in colorectal cancer[14-16]. KAI1 has been thought to be one of such metastasis suppressor genes, because it was shown to suppress the ability of human prostatic cancer cells to metastasize when the tumor was transplanted into nude mice[1] and because KAI1 mRNA expression was reduced in advanced pancreatic cancer[17] so that the pancreatic cancer cells spread to lymph nodes and distant organs. Furthermore, the transfer of the KAI1 gene into mammary cancer cells has been shown to lead to suppression of their metastatic potential, whereas their primary tumor growth has not been affected[18-20].
KAI1 is a member of the transmembrane-4 superfamily (TM4SF), many of which, including KAI1, are CD antigens present on the surface of leukocytes[21,22]. At least three TM4SF members are implicated in metastasis, including CD9/MRP-1, CD63/ME491, and CD82/KAI1[23]. KAI1 and other TM4SF members, such as integrins and E-cadherin, have been demonstrated to bind to each other and relay extracellular signals to signal transduction pathways that are important in cellular adhesion, invasive motility, and metastasis[24-27].
In our previous studies, we transfected the KAI1 cDNA into a colorectal carcinoma cell line, LoVo, and found that the KAI1 transfectant exhibited significantly increased homotypic cell adhesion and suppressed invasion and metastasis[9]. Our findings were consistent with those in the studies of prostatic cancer[2] and breast cancer[18] cells.
In this study, we first investigated KAI1 mRNA expression in 4 colorectal carcinoma cell lines and found KAI1 gene expression was much higher in HT29 and SW480 cell lines than in SW620 and LoVo cell lines. SW480 was isolated from a high-grade primary colonic tumor, and SW620 was isolated from a metastatic lymph node from the same patient 1 year later at the time of clinical relapse. KAI1 protein expression was high in SW480 but reduced in SW620. The colonic cancer cell line derived from metastatic lesions, LoVo, hardly exhibited any expression. In our observation we found that KAI1 gene expressions both at mRNA and protein level, were inversely correlated with the metastatic potential of some established colorectal carcinoma cell lines.
We next examined KAI1 expression in normal colonic mucosa, carcinoma and lymphatic metastasis by in situ hybridization. The results indicated that KAI1 gene was highly expressed in colonic carcinoma compared with normal colonic epithelium and lymphatic metastasis. We found that although the expression had no relationship with histological grades, it was significantly correlated with Dukes stages. This indicates that the KAI1 mRNA expression may be positively related with the Dukes stages. The data shown in Table 1 demonstrate that KAI1 expression increases at an earlier tumor stage of colonic carcinoma, while decreases at advanced stages, and is possibly lost in metastases. That is to say, the expression of KAI1 has a reverse correlation with metastasis in colorectal carcinoma.
How does reduced TM4SF expression cause changes of invasive ability of tumor cells It was hypothesized that tetraspanins might be implicated in the assembly of integrin-contained signaling complexes, thus modulating the function of integrin receptors in cell migration[24]. Some results indicated that integrin-tetraspanin protein complexes played an important role in regulating protrusive activity of the tumor cells and contributed to extracellular matrix-induced production of matrix metalloproteinase 2 (MMP-2), and as a consequence, the invasive ability of cells[28].
KAI1 has been extensively studied for its involvement in the progression of different human cancers. The mechanism of down-regulation of KAI1 has also been analyzed, however, much debate still exists. Recent study found a putative p53 consensus-binding site within the promoter region of KAI1 and demonstrated that the loss of p53 function, which was commonly observed in many types of cancer, led to the down-regulation of the KAI1 gene, which might result in the progression of metastasis[29]. But other data suggested that the down- regulation of KAI1 was not associated with either mutation, allelic loss, methylation of the promoter, or p53 regulation[30]. Our previous study also demonstrated that mutation of the KAI1 gene, methylation of CpG islands and the abnormality of p53 were not related to low expression of KAI1.
In conclusion, KAI1 expression increases in an earlier tumor stage of colonic carcinoma, decreases in advanced stages, and is possibly lost in lymphatic metastases. The loss of KAI1 expression is correlated with higher stage, a surrogate marker for metastatic potential. The down-regulation of KAI1 in Dukes C and loss in metastasis demonstrate that loss of KAI1 expression occurs in cancer progression. The selection of cells that have the ability to spread from the primary tumor to the metastasis may favor those cells that have lost KAI1 expression. Those cells would be expected to be less adhesive, more invasive, and more motile[31], and these characteristics are necessary for metastasis.
Supported by the National Natural Science Foundation, No. 30370649
Edited by Kumar M Proofread by Chen WW and Xu FM
Dong JT, Lamb PW, Rinker-Schaeffer CW, Vukanovic J, Ichikawa T, Isaacs JT, Barrett JC. KAI1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2. Science. 1995;268:884-886.[PubMed] [DOI]
Dong JT, Isaacs WB, Barrett JC, Isaacs JT. Genomic organization of the human KAI1 metastasis-suppressor gene. Genomics. 1997;41:25-32.[PubMed] [DOI]
Liu FS, Chen JT, Dong JT, Hsieh YT, Lin AJ, Ho ES, Hung MJ, Lu CH. KAI1 metastasis suppressor gene is frequently down-regulated in cervical carcinoma. Am J Pathol. 2001;159:1629-1634.[PubMed] [DOI]
Debies MT, Welch DR. Genetic basis of human breast cancer metastasis. J Mammary Gland Biol Neoplasia. 2001;6:441-451.[PubMed] [DOI]
Friess H, Guo XZ, Tempia-Caliera AA, Fukuda A, Martignoni ME, Zimmermann A, Korc M, Büchler MW. Differential expression of metastasis-associated genes in papilla of vater and pancreatic cancer correlates with disease stage. J Clin Oncol. 2001;19:2422-2432.[PubMed]
Miyazaki T, Kato H, Shitara Y, Yoshikawa M, Tajima K, Masuda N, Shouji H, Tsukada K, Nakajima T, Kuwano H. Mutation and expression of the metastasis suppressor gene KAI1 in esophageal squamous cell carcinoma. Cancer. 2000;89:955-962.[PubMed] [DOI]
Jackson P, Kingsley EA, Russell PJ. Inverse correlation between KAI1 mRNA levels and invasive behaviour in bladder cancer cell lines. Cancer Lett. 2000;156:9-17.[PubMed] [DOI]
Liu FS, Dong JT, Chen JT, Hsieh YT, Ho ES, Hung MJ. Frequent down-regulation and lack of mutation of the KAI1 metastasis suppressor gene in epithelial ovarian carcinoma. Gynecol Oncol. 2000;78:10-15.[PubMed] [DOI]
Liu L, Wu DH, Li ZG, Yang GZ, Ding YQ. Effects of KAI1/CD82 on biological behavior of human colorectal carcinoma cell line. World J Gastroenterol. 2003;9:1231-1236.[PubMed]
Wu DH, Liu L, Chen LH, Ding YQ. Expression of KAI1/CD82 in human colorectal tumor. Di Yi Jun Yi Da Xue Xue Bao. 2003;23:714-715, 719.[PubMed]
Dursun A, Akyürek N, Günel N, Yamaç D. Prognostic implication of nm23-H1 expression in colorectal carcinomas. Pathology. 2002;34:427-432.[PubMed] [DOI]
El-Bahrawy MA, Poulsom R, Jeffery R, Talbot I, Alison MR. The expression of E-cadherin and catenins in sporadic colorectal carcinoma. Hum Pathol. 2001;32:1216-1224.[PubMed] [DOI]
Wong K, Rubenthiran U, Jothy S. Motility of colon cancer cells: modulation by CD44 isoform expression. Exp Mol Pathol. 2003;75:124-130.[PubMed] [DOI]
Forte A, D'Urso A, Gallinaro LS, Lo Storto G, Soda G, Bosco D, Bezzi M, Vietri F, Beltrami V. NM23 expression as prognostic factor in colorectal carcinoma. G Chir. 2002;23:61-63.[PubMed]
Heys SD, Langlois N, Smith IC, Walker LG, Eremin O. NM23 gene product expression does not predict lymph node metastases or survival in young patients with colorectal cancer. Oncol Rep. 1998;5:735-739.[PubMed]
Cheah PY, Cao X, Eu KW, Seow-Choen F. NM23-H1 immunostaining is inversely associated with tumour staging but not overall survival or disease recurrence in colorectal carcinomas. Br J Cancer. 1998;77:1164-1168.[PubMed] [DOI]
Guo X, Friess H, Graber HU, Kashiwagi M, Zimmermann A, Korc M, Büchler MW. KAI1 expression is up-regulated in early pancreatic cancer and decreased in the presence of metastases. Cancer Res. 1996;56:4876-4880.[PubMed]
Yang X, Wei LL, Tang C, Slack R, Mueller S, Lippman ME. Overexpression of KAI1 suppresses in vitro invasiveness and in vivo metastasis in breast cancer cells. Cancer Res. 2001;61:5284-5288.[PubMed]
Ono M, Handa K, Withers DA, Hakomori S. Motility inhibition and apoptosis are induced by metastasis-suppressing gene product CD82 and its analogue CD9, with concurrent glycosylation. Cancer Res. 1999;59:2335-2339.[PubMed]
Takaoka A, Hinoda Y, Sato S, Itoh F, Adachi M, Hareyama M, Imai K. Reduced invasive and metastatic potentials of KAI1-transfected melanoma cells. Jpn J Cancer Res. 1998;89:397-404.[PubMed] [DOI]
White A, Lamb PW, Barrett JC. Frequent downregulation of the KAI1(CD82) metastasis suppressor protein in human cancer cell lines. Oncogene. 1998;16:3143-3149.[PubMed] [DOI]
Okochi H, Mine T, Nashiro K, Suzuki J, Fujita T, Furue M. Expression of tetraspans transmembrane family in the epithelium of the gastrointestinal tract. J Clin Gastroenterol. 1999;29:63-67.[PubMed] [DOI]
Adachi M, Taki T, Konishi T, Huang CI, Higashiyama M, Miyake M. Novel staging protocol for non-small-cell lung cancers according to MRP-1/CD9 and KAI1/CD82 gene expression. J Clin Oncol. 1998;16:1397-1406.[PubMed]
Rubinstein E, Le Naour F, Lagaudrière-Gesbert C, Billard M, Conjeaud H, Boucheix C. CD9, CD63, CD81, and CD82 are components of a surface tetraspan network connected to HLA-DR and VLA integrins. Eur J Immunol. 1996;26:2657-2665.[PubMed] [DOI]
Bienstock RJ, Barrett JC. KAI1, a prostate metastasis suppressor: prediction of solvated structure and interactions with binding partners; integrins, cadherins, and cell-surface receptor proteins. Mol Carcinog. 2001;32:139-153.[PubMed] [DOI]
Hashida H, Takabayashi A, Tokuhara T, Taki T, Kondo K, Kohno N, Yamaoka Y, Miyake M. Integrin alpha3 expression as a prognostic factor in colon cancer: association with MRP-1/CD9 and KAI1/CD82. Int J Cancer. 2002;97:518-525.[PubMed] [DOI]
Hemler ME, Mannion BA, Berditchevski F. Association of TM4SF proteins with integrins: relevance to cancer. Biochim Biophys Acta. 1996;1287:67-71.[PubMed] [DOI]
Muneyuki T, Watanabe M, Yamanaka M, Shiraishi T, Isaji S. KAI1/CD82 expression as a prognosic factor in sporadic colorectal cancer. Anticancer Res. 2001;21:3581-3587.[PubMed]
Mashimo T, Watabe M, Hirota S, Hosobe S, Miura K, Tegtmeyer PJ, Rinker-Shaeffer CW, Watabe K. The expression of the KAI1 gene, a tumor metastasis suppressor, is directly activated by p53. Proc Natl Acad Sci USA. 1998;95:11307-11311.[PubMed] [DOI]
Uzawa K, Ono K, Suzuki H, Tanaka C, Yakushiji T, Yamamoto N, Yokoe H, Tanzawa H. High prevalence of decreased expression of KAI1 metastasis suppressor in human oral carcinogenesis. Clin Cancer Res. 2002;8:828-835.[PubMed]
Geradts J, Maynard R, Birrer MJ, Hendricks D, Abbondanzo SL, Fong KM, Barrett JC, Lombardi DP. Frequent loss of KAI1 expression in squamous and lymphoid neoplasms. An immunohistochemical study of archival tissues. Am J Pathol. 1999;154:1665-1671.[PubMed] [DOI]