[CANCER RESEARCH 61, 7943–7949, November 1, 2001] Inactivation of Human SRBC, Located within the 11p15.5-p15.4 Tumor Suppressor Region, in Breast and Lung Cancers1 Xie L. Xu, Leeju C. Wu, Fenghe Du, Arthur Davis, Michael Peyton, Yoshio Tomizawa, Anirban Maitra, Gail Tomlinson, Adi F. Gazdar, Bernard E. Weissman, Anne M. Bowcock, Richard Baer, and John D. Minna2 Hamon Center for Therapeutic Oncology Research [X. L. X., M. P., Y. T., A. M., G. T., A. F. G., J. D. M.], and Department of Biochemistry [L. C. W.], University of Texas Southwestern Medical Center, Dallas, Texas 75390; Institute of Cancer Genetics, Columbia University College of Physicians and Surgeons New York, New York 10032 [R. B.]; Division of Human Genetics, Department of Genetics, Pediatrics and Medicine, Washington University School of Medicine, St. Louis, Missouri 63110 [F. D., A. M. B.]; Department of Pathology and Laboratory Medicine and The Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599 [A. D., B. E. W.] ABSTRACT 3). Region 1 extends from D11S1318 to D11S4088, overlapping with the previous identified LOH regions in breast cancer (2, 17), ovarian A cDNA clone encoding human SRBC [serum deprivation response carcinoma (7), Wilms’ tumor (14), rhabdomyosarcoma (14, 15), and factor (sdr)-related gene product that binds to c-kinase] was isolated in a gastric adenocarcinoma (9). Region 2 is defined by D11S1338 and yeast two-hybrid screening, with amino acids 1–304 of BRCA1 as the probe. The human SRBC gene (hSRBC) was mapped to chromosome D11S1323, which overlaps with LOH regions described for breast region 11p15.5-p15.4, close to marker D11S1323, at which frequent loss of cancer (18), NSCLC (5), and Wilms’ tumor (11). Region 1 also heterozygosity (LOH) has been observed in sporadic breast, lung, ovarian, overlaps with a locus that contains several imprinted genes such as and other types of adult cancers as well as childhood tumors. hSRBC- H19, IGF2, INS, TH, HASH2, KVLQT1, and p57Kip2 (Fig. 1A), some coding region mutations including frame shift and truncation mutations of which are implicated in a variety of cancers (19). The breakpoints were detected in a few ovarian and lung cancer cell lines. More signifi- of chromosome translocation and inversion associated with malignant cantly, the expression of hSRBC protein was down-regulated in a large rhabdoid tumors and Beckwith-Wiedemann syndrome are also within fraction [30 (70%) of 43] of breast, lung, and ovarian cancer cell lines, this region (20). Region 2 is more centromeric (Fig. 1A) and LOH in whereas strong expression of hSRBC protein was detected in normal Region 2 is associated with clinical parameters of more aggressive mammary and lung epithelial cells. The down-regulation of hSRBC ex- breast tumors and a poorer prognostic indicators, such as aneuploidy, pression in cancer cells was associated with hypermethylation of CpG dinucleotides in its promoter region, and 3 (60%) of 5 primary breast high S-phase fraction, and the presence of metastasis in regional tumors and 11 (79%) of 14 primary lung tumors were also found to be lymph nodes (3). Similarly, 11p allele loss is also observed in lung hypermethylated. Treatment of breast cancer MCF7 cells with 5؅azacyti- cancer at a frequency ranging between 11 and 50% (4, 5). LOH at 11p dine and Trichostatin A resulted in expression of hSRBC, confirming in lung cancer is correlated with advanced T stage and nodal involve- DNA methylation as the mode of inactivation. Our results suggest that ment in NSCLC (21). O’Brant and Bepler (22) have mapped two epigenetic or mutational inactivation of hSRBC may contribute to the regions (LOH11A and LOH11B) on chromosome 11p15.5 that have pathogenesis of several types of human cancers, marking hSRBC as a frequent allele loss in lung cancer. LOH11A is centromeric between candidate tumor suppressor gene. loci D11S1758 and D11S12, and LOH11B is telomeric between HRAS and D11S1363. INTRODUCTION Nonrandom chromosome deletion and allele loss often marks the Allelic loss at the chromosome segment 11p15.5 is frequently site of inactivation of TSGs residing in a particular chromosomal observed in a variety of adult solid tumors including breast (1–3), lung region. Thus, the finding of frequent LOH in breast and lung cancer (4, 5), ovarian (6, 7), bladder (8), stomach (9), and adrenal cortical strongly suggests the presence of one or several 11p TSGs. The (10) cancers as well as some childhood tumors including Wilms’ observation that physical transfer of 11p chromosomal fragments into tumor (11–13), rhabdomyosarcoma (14, 15), and hepatoblastoma tumor cell lines was able to reverse the tumorigenic phenotype of (16). In breast cancer, the frequency of LOH3 for 11p15.5 detected in cancer cells provided additional functional evidence for the existence invasive ductal breast cancers is around 30–60% (1, 2). Two distinct of TSGs in this region (23–25). Because of the complex LOH pattern regions on chromosome 11p15 that are subjected to LOH in breast in this region and the lack of homozygous deletions to help target a cancer have been identified and refined by Karnik et al. (Fig. 1A; Ref. positional cloning effort, it has been hard to identify candidate TSG(s) for this chromosome region. In this study, we described such a candidate, the hSRBC gene. The gene was isolated in a two-hybrid Received 3/26/01; accepted 8/27/01. The costs of publication of this article were defrayed in part by the payment of page screen for proteins interacting with the product of the breast cancer charges. This article must therefore be hereby marked advertisement in accordance with susceptibility gene BRCA1. RH mapping localized hSRBC gene to 18 U.S.C. Section 1734 solely to indicate this fact. chromosome region 11p15.5 between D11S1323 and D11S1338. 1 Supported by National Cancer Institute Lung Cancer Specialized Programs of Re- search Excellence (SPORE) Grant P50 CA70907 (to J. D. M.), G. Harold and Leila Y. Moreover, several mutations including frame shift and truncation Mathers Charitable Foundation (to J. D. M.), Cancer Research Foundation of North Texas mutations were identified in ovarian and lung cancer cell lines. Most (to J. D. M.), and NIH Grants RO1-CA76334 (to R. B.) and -CA63176 (to B. E. W.). X. L. X. was a recipient of a Postdoctoral Fellowship Award from Susan G. Komen Breast significantly, the expression of hSRBC mRNA and protein was down- Cancer Foundation, and M.P. of Department of Defense Grant DAMD17-94-J-4077. regulated in a large fraction of breast and lung cancer cell lines, and 2 To whom requests for reprints should be addressed, at Hamon Center for Therapeutic the down-regulation is associated with hypermethylation in the Oncology Research, University of Texas Southwestern Medical Center-Dallas, 6000 Harry Hines Boulevard, Dallas, Texas 75390-8593. Phone: (214) 648-4900; Fax: (214) hSRBC promoter region. The evidence provided here suggests that 648-4940; E-mail: [email protected]. inactivation of hSRBC may contribute to the pathogenesis of several 3 The abbreviations used are: LOH, loss of heterozygosity; BAC, bacterial artificial types of cancer. chromosome; HMEC, normal human mammary epithelial cell; NHBE, normal human bronchial epithelial (cell); NSCLC, non-SCLC; PKC, protein kinase C; PTRF, RNA- polymerase I-transcript release factor; RH, radiation hybrid; SAEC, normal human lung MATERIALS AND METHODS small airway epithelial cells; SCLC, small cell lung cancer; SRBC, SDPR (sdr)-related gene product that binds to c-kinase; TSA, Trichostatin A; TSG, tumor suppressor gene; Cell Lines, Tissues, Genomic DNAs, RNAs, and Protein Expression UT Southwestern, University of Texas Southwestern Medical Center; NCBI, National Center for Biotechnology; mCpG, methylated CpG; SDPR, serum deprivation response Studies. All of the breast and lung cancer cell lines were from the Hamon (factor); hSRBC, human SRBC; SHGC, Stanford Human Genome Center. Center collection (UT Southwestern) generated by the authors and were 7943 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2001 American Association for Cancer Research. INACTIVATION OF hSRBC IN BREAST AND LUNG CANCER by 30 cycles of 94°C (30 s), 65°C (30 s), and 72°C (30 s), with a final extension at 72°C for 7 min]. The primers for D11S1323 are: forward, 5Ј-TCTAATTTCTTCTCCCACCC-3Ј, and reverse, 5Ј-TGGGAGTTTCTCT- GTGCTAG-3Ј. The primers for D11S1338 are: forward, 5Ј-TCAGAAATCT- GATGGAAAAGTC-3Ј, and reverse, 5Ј-TGCTACTTATTTGGAGTGTGAA- 3Ј. The cycle conditions for D11S1323- and D11S1338-specific primers were 68°C-58°C touchdown [one cycle at 95°C (5 min); 10 touchdown cycles at 94°C (30 s), 68°C (30 s) with a decrease of 1°C each cycle and at 72°C for 30 s; followed by 30 cycles of 94°C (30 s), 58°C(30 s) and 72°C (30 s), with a final extension at 72°C for 7 min]. RH Mapping. Mapping of the chromosome location of the hSRBC gene was performed using Stanford TNG RH panel (Research Genetics). The PCR reaction for hSRBC was performed as described above for the BAC DNA. The RH raw data from these tests are: 0100000100 000000R100 0000000000 1000110001 0001000000 1000010010 0001001001 0000000000 0100000010. Mutational Analysis. The primer pairs of hSRBC gene for PCR amplifi- cation from 100 ng of genomic DNA are: 5Ј-GGAGCAGAGCGGTCAGG- GATC-3Ј and 5Ј-GGACCGTTTGAGGTCACTGAC-3Ј for exon 1; and 5Ј-GCTGTGTCCGTCACATGCAG-3Ј and 5Ј-AGGCAGGCAACACCAG- CCC-3Ј for exon 2. The PCR reaction was performed as described above (75°C-65°C touchdown). The PCR products were purified with Qiagen PCR purification kit and sequenced with ABI 377 automatic DNA sequencer. Bisulfite Genomic Sequencing. Genomic DNA (1 ␮g) was treated with sodium bisulfite as described previously (30). Forty ng of treated DNA was used as template in PCR amplification reaction in a volume of 25 ␮l.