[CANCER RESEARCH 61, 4942– 4946, July 1, 2001] Advances in Brief Mutations of Tumor Necrosis Factor-related Apoptosis-inducing Ligand Receptor 1 (TRAIL-R1) and Receptor 2 (TRAIL-R2) Genes in Metastatic Breast Cancers 1 Min Sun Shin, Hong Sug Kim, Sug Hyung Lee, Won Sang Park, Su Young Kim, Jik Young Park, Jong Heun Lee, Sang Kyu Lee, Shi Nae Lee, Sang Seol Jung, Ji Youn Han, Hyang Kim, Jung Young Lee, and Nam Jin Yoo 2 Departments of Pathology [M. S. S., H. S. K., S. H. L., W. S. P., S. Y. K., J. Y. P., J. H. L., S. K. L., S. N. L., J. Y. L., N. J. Y.], Surgery [S. S. J.], and Internal Medicine [J. Y. H.], College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea, and Department of Internal Medicine, Sungkyunkwan University School of Medicine, Seoul 110- 102, Korea [H. K.] Abstract Several lines of evidence suggest that apoptosis dysregulation plays an important role in cancer metastasis. In this study, to explore the possibility that the mutations of death receptors are involved in the metastasis mechanism, we analyzed the death domains of Fas and tumor necrosis factor-related apoptosis-inducing ligand-receptor 1 and -2 (TRAIL-R1 and -R2) genes for the detection of somatic mutations in 57 breast cancers with (n  34) or without (n  23) metastasis to the regional lymph nodes. We found seven mutations (three TRAIL-R1 and four TRAIL-R2 mutations), and these mutations were detected only in the breast cancers with metas- tasis. Furthermore, we also analyzed the allelic losses of chromosome 8p21–22, where TRAIL-R1 and R2 reside in the same series of breast cancers, and found that the allelic losses were significantly higher in metastatic breast cancers. We expressed the tumor-derived TRAIL-R1 and TRAIL-R2 mutants in 293 cells and found that apoptosis was suppressed. These data suggest that TRAIL-R1 and R2 genes are relevant to the frequent loss of chromosome 8p21–22 in breast cancer and that the inacti- vating mutations of TRAIL-R1 and -R2 genes play a role in the metastasis of breast cancer. Introduction It is now believed that clonal expansion and tumor growth is the result of the deregulation of intrinsic proliferation (cell division) and cell death (apoptosis; Ref. 1). Aberrant cell survival resulting from the inhibition of apoptosis is expected to contribute to the development and progression of cancers, and cancer cells would gain a selective growth advantage by blocking the apoptosis (1). Metastasis is a highly complex process involving the survival of cancer cells, both in the blood stream and within specific organs. Recent findings showed that metastatic cancer cells exhibit a higher resistance to apoptosis com- pared with their poorly-metastatic counterparts (2), which indicates that metastatic cancer cells may have many more dysregulations in the apoptosis pathway. However, there have been few experimental data linking apoptosis and metastasis (3, 4). Death factor/death receptor-induced cell death is one of the main apoptosis pathways (1). Death receptors such as Fas, TRAIL-R1 3 and TRAIL-R2 transmit a death signal on binding with their natural ligands (death factors; Refs. 1, 5–7). However, many types of cancer cells are resistant to death factor-induced apoptosis (1). Fas ligand and TRAIL-induced apoptosis can be blocked by several mechanisms in cancer cells, one of which is the mutation of the primary structures of Fas and TRAIL-R2 (8 –10). Interestingly, Fas and TRAIL-R2 muta- tions have been detected in several types of human cancers with frequent allelic losses of chromosomes 10q24 and 8p21–22, where Fas and TRAIL-R2 reside, respectively (8 –10). These data indicate that chromosomes 10q24 and 8p21–22 may harbor one or more tumor suppressor genes and suggest that Fas and TRAIL-R2 might be the candidate tumor suppressor genes in these loci. Furthermore, because TRAIL-R1 gene also exists in chromosome 8p21–22, it might be another candidate tumor suppressor gene in chromosome 8p21–22 (11). Breast cancer has also shown frequent allelic losses of 10q24 and 8p21–22 (12, 13), but the mutational studies of Fas, TRAIL-R1, and TRAIL-R2 genes in breast cancer have not yet been reported. There is evidence that death receptor mutation is involved in the pathogenesis of human cancer metastasis (4). In one adult T-cell leukemia patient, Fas gene mutation was detected in metastatic cancer cells but not in primary cancers, and the metastatic cancer cells were resistant to agonistic Fas antibody treatment, but the primary cancer cells were not (4). Therefore, it can be hypothesized that other members of the death receptor are mutated in some cancers and such mutations might be involved in the metastasis mechanisms of the cancers. Also, there are reports that allelic losses of chromosomes 10q24 and 8p21–22 of breast cancer occur during the progression of the tumors (12, 14, 15). In the present study, to explore the possibilities that Fas, TRAIL-R1, and TRAIL-R2 genes are relevant to the frequent allelic losses at chromosomes 10q24 and 8p21–22 in breast cancers and that these potential mutations show different incidences between nonmetastatic and metastatic breast cancers, we analyzed a series of 57 IDCs of the breast for the detection of genetic alteration of the death domains of Fas, TRAIL-R1, and TRAIL-R2 genes. Materials and Methods Tissue Samples. Formalin-fixed and paraffin-embedded tissues of human IDC of breast with (n = 34) or without (n = 23) regional lymph node metastasis were obtained from 57 surgically treated patients. The stage was determined according to the American Joint Committee on Cancer (16). The cases with regional lymph node metastasis consisted of 9 stage IIA, 19 stage IIB, 4 stage IIIA, and 2 stage IIIB IDCs, and the cases without regional lymph node metastasis consisted of 11 stage I, 10 stage IIA, and 2 stage IIB IDCs. Microdissection and DNA Extraction. Malignant cells were selectively procured from H&E-stained slides without any normal cell contamination using a 30-gauge 1 /2-inch hypodermic needle (Becton Dickinson, Franklin Lakes, NJ) affixed to a micromanipulator, as described previously (17). We also microdissected infiltrating lymphocytes for corresponding normal DNA from the same slide in all of the cases. DNA extraction was performed by a modified single-step DNA extraction method, as described previously (17). SSCP Analysis. Genomic DNA, each from tumor cells or corresponding normal cells was amplified with 3 primer pairs for the Fas gene, 2 primer pairs Received 2/7/00; accepted 5/14/01. 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. 1 Supported by Grant 1999-2-207-002-5) from Basic Research Program of the Korean Science and Engineering Foundation. 2 To whom requests for reprints should be addressed, at Department of Pathology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea. Phone: 82-2-590-1191; Fax: 82-2-537-6586. 3 The abbreviations used are: TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; TRAIL-R1, TRAIL-receptor 1; TRAIL-R2, TRAIL-receptor 2; IDC, invasive ductal carcinoma; SSCP, single-strand conformation polymorphism; LOH, loss of het- erozygosity; GFP, green fluorescence protein; DAPI, 4'-6-diamidino-2-phenylindole. 4942 Research. on November 28, 2021. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from