Inactivation of Human SRBC, Located Within the 11P15.5-P15.4 Tumor Suppressor Region, in Breast and Lung Cancers1

[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

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 amplification 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. The primer pairs for PCR amplification are: forward, 5Ј-ATTTTTATTGGTGT- Fig. 1. The hSRBC gene is located at 11p15.5-p15.4 on the same BAC clone as GGGAGG-3Ј, and reverse, 5Ј-CCCTACAAACCCTCTCACTCT-3Ј. The PCR D11S1323. A, schematic map of the chromosome location of the hSRBC gene. The order ϫ reaction was performed in 1 Hot Star Taq buffer containing 2.5 mM MgCl2, of the markers on the schematic map is mostly based on the SHGC G3 RH map as well 500 nM primers, 160 ␮M dNTP, and 1 unit of Hot Star Taq (Qiagen). The cycle as information from the NCBI GB4 map, Genethon Genetic map, and Genome Database map. Bold, the distance between backbone markers are centirays for 10,000-rad dose conditions were: one cycle at 94°C (12 min); 2 touchdown cycles at 94°C (30 (cR10,000s); highlighted region, the imprinted locus; various boxes on the right, the LOH s), 61°C (45 s) with a decrease of 1°C each cycle, and at 72°C for 30 s; regions reported previously for breast, Wilms’ tumor (WT) and NSCLC. B, hSRBC gene followed by 35 cycles of 94°C (30 s), 60°C(30 s) and 72°C (30 s), with a final and marker D11S1323 (but not D11S1338) reside on the same BAC clone (368H20 from extension at 72°C for 7 min. A second PCR was performed using 0.5 ␮lofthe Research Genetics). PCR amplification was performed using hSRBC-specific (left), D11S1323-specific (middle), or D11S1338-specific (right) primers, with BAC clone first reaction with the same conditions. The PCR products were purified with 368H20 DNA as the template (Lanes 2, 6, and 10). As a positive control for the PCR Qiagen PCR purification kit, and sequenced with an ABI 377 automatic DNA reaction, the whole genomic DNA from MCF7 cells was used in parallel reaction (Lanes sequencer. and 11). No template was added in the negative control reaction (Lanes 4, 8, and 12). Treatment of MCF7 Cells with 5؅-Azacytidine and TSA. MCF7 cells ,7 ,3 M, molecular weight marker. References (RE) are for breast (Ref. 3), Wilms’ tumor Ј (Ref. 11), and NSCLC (Refs. 5 and 22). grown to 30% confluency in 24-well plates were treated with 5 -azacytidine (Sigma Chemical Co.) at different concentrations for 2 days. Then, different concentrations of TSA were added, and cells were incubated for 2 more days cultured with RPMI 1640 containing 5% fetal bovine serum. Most are also before harvest. On harvest, cells were lysed directly in SDS-PAGE loading available from American Type Culture Collection (Chantilly, VA). Ovarian buffer, and the expression of hSRBC was determined by immunoblotting with cancer cell line 2008 was provided by Dr. C. Muller (UT Southwestern) and the hSRBC monoclonal antibody. cultured in DMEM with 10% fetal bovine serum. All of the genomic DNAs and total RNAs of the cultured cell lines were prepared as described previously RESULTS (26). HMECs, normal human bronchial/tracheal epithelial cells (cultured with retinoic acid), and normal human lung SAECs were purchased from Clonetics Human Orthologue of Rat SRBC (hSRBC) and Its Chromo- Corporation, cultured as recommended, and analyzed at passages 7–10. For- some Location. In an effort to study the BRCA1-mediated tumor malin-fixed, paraffin-embedded primary lung and breast carcinoma specimens suppression pathway, a cDNA clone was isolated when a normal were retrieved from the surgical pathology archives of UT Southwestern- mammary epithelial cDNA library was screened by the yeast two- affiliated hospitals, microdissected using a laser capture microscope (27), and hybrid method, using a recombinant protein containing amino acids DNA extracted as described previously (28). Northern blotting and Western 1–304 of BRCA1 as the “bait” (31). This was performed by L. C. W. immunoblotting were performed as described previously (29). Twenty ␮gof total RNA prepared from the indicated cell lines were electrophoresed, trans- in R. B.’s laboratory at UT Southwestern. The cDNA contains an ferred, and hybridized with a full open reading frame cDNA probe for hSRBC open reading frame of 261 amino acids (Accession no. AF339881). using standard procedures. The monoclonal antibody against hSRBC was BLAST search revealed the cDNA encodes the human orthologue of generated by immunizing Balb/C mice with a glutathione S-transferase fusion the rat SRBC gene that was isolated in a far-Western approach using protein containing amino acids 201–261 of hSRBC by standard fusion and PKC-␦ isoform (PKC-␦) as the probe (32). Thus, we refer to the screening procedures. human orthologue as hSRBC. hSRBC is also identical to the “un- BAC DNA Isolation, PCR, and Sequencing. BAC clone 368 H20 was known” NH2-terminal cellular fusion protein in one form of activated obtained from Research Genetics, maintained as recommended, and DNA c-Raf-1 (X06409; Ref. 33). hSRBC is 81% identical to its rat ortho- isolated using a Midi plasmid kit (Qiagen). The PCR reaction was performed logue at the amino acid level, and, like rat SRBC, the amino acid with Amplitaq Gold (Perkin-Elmer) in 1ϫ buffer containing 1.5 mM MgCl , 2 sequence of hSRBC reveals a leucine zipper-like motif in its NH - 5% DMSO, 200 ␮M dNTP, 0.2 mM primers, and 50 ng of BAC DNA in a 2 20-␮l reaction. The primers for detecting hSRBC are: forward, 5Ј-GGAGCA- terminal region. As was reported previously, SRBC is also homolo- GAGCGGTCAGGGATC-3Ј, and reverse, 5Ј-GGACCGTTTGAGGTCACT- gous to several molecules in the database: both human and mouse GAC-3Ј; and the cycle conditions for hSRBC-specific primers were 75°C-65°C SDPR factor (AF085481 and S67386, respectively; Ref. 34) and touchdown [one cycle at 95°C (5 min); 10 touchdown cycles at 94°C (30 s), PTRF (AF036249; Ref. 35), and a putative leucine-zipper protein that 75°C (30 s) with a decrease of 1°C each cycle, and at 72°C for 30 s; followed appears to be the chicken orthologue of PTRF (D82079 and D26315). 7944

A BLAST search of the NCBI expressed sequence tag database Table 1 hSRBC mutations and polymorphisms found many expressed sequence tags matched to the sequence of Cell Wild-type hSRBC gene, one of which (AA030008) has already been placed at Mutations type Alterations Codon Predicted effect allele present chromosome 11p15.5-p15.4 on the GeneBridge 4 RH map of NCBI Cell lines a GeneMap 99.4 To confirm the chromosomal location of hSRBC,we 2008 Ovary 89delG 18 frameshift Yes NCI-H358 NSCLC C604T 190 R to stop Yesa performed RH mapping using the Stanford TNG RH panel (Research NCI-H510 SCLC 177del 21 bp 48–55 deletion of Yesa Genetics), which gives higher resolution than GB4 and G3 panel. The (RRQGGLA) hSRBC gene was shown to be closely linked to SHGC-2070 (Genome NCI-H2081 SCLC C611G 192 A to G Yes Database locus D11S1323) at 11p15.5 on the SHGC G3 map,5 with a Predicted Frequencyb Alterations Codon effect (%) log of odds (LOD) score of 12.48 and the distance between hSRBC ϳ Polymorphisms C59G 8 P to R 8 and D11S1323 of 13 centirays at 50,000 rads (cR50,000; 26–39 kb). G378A 114 Silent 8 Further analysis using the RH raw data of telomeric marker SHGC- T509C 158 L to P 45 14206 (RHdb: RH95600) and centromeric marker SHGC-30684 T690A 218 Silent 67 C799T 255 L to F 5 (RHdb: RH96895) suggested that the hSRBC gene is located centro- a Immunoblotting indicates these tumor cell lines do not express wild-type hSRBC meric to D11S1323, thus residing between D11S1323 and D11S1338 protein despite the presence of a wild-type hSRBC allele (Fig. 2E). H2081 has not been (Fig. 1A), right within the LOH Region 2 as described by Karnik et al. tested in immunoblotting. b (3), and very close to the LOH region LOH11A in lung cancer as The allele frequencies were determined from 53 normal Caucasian individuals. described by O’Briant and Bepler (22). Moreover, a specific DNA fragment could be amplified with hSRBC-, or D11S1323-specific primer pairs from the same BAC clone (368 H20 from Research the homologous proteins. None of the four alterations are present in 60 Genetics; Fig. 1B), confirming that they are less than 200 kb apart. normal control DNAs and 134 other cancer cell lines or tumor DNAs This BAC clone was partially sequenced providing 7466 bp of (total 388 chromosomes tested). However, we cannot formally ex- genomic DNA (AF408198). clude that they represent rare polymorphisms, because no matched Mutational Study of the hSRBC Gene. Comparison of the cDNA normal samples were available to allow us to directly determine sequence of hSRBC with its corresponding genomic DNA sequence, whether the alterations are tumor-acquired. All of the alterations were which was obtained by sequence analysis of BAC clone 368 H20 confirmed with multiple PCR amplification and sequence analysis (Research Genetics), revealed that the coding region of hSRBC gene using two batches of independent DNA preparations. is organized into two exons: exon 1 covers codons 1–128, and exon 2 Down-Regulation of hSRBC Expression in Breast and Lung contains codons 129–261. The two exons are separated by a small Cancer Cell Lines. The hSRBC protein is expressed in both HMEC, intron of 529 bp. Also PCR-single-strand-conformation-polymor- and NHBE cell cultures as well as in a BRCA1 mutant breast cancer phism analysis of genomic DNAs from 53 normal Caucasian individ- cell line HCC1937 (Fig. 2A), as detected by immunoblot (37). In uals identified five common polymorphisms in the hSRBC coding addition, hSRBC mRNA was also detected in NHBE cells and SAEC sequence (Table 1). Because the hSRBC gene was mapped to a region (Fig. 2B). By contrast, loss of hSRBC protein expression was observed that shows frequent LOH in multiple types of human cancers includ- in 4 of 11 breast (Fig. 2C) and 26 of 32 lung cancer cell lines (Fig. ing breast and lung, we first examined LOH within the hSRBC coding 2D). In contrast to the conventional two-hit model, in those tumor cell region in lung cancer cell lines with single-strand conformation poly- lines in which a hSRBC mutation was detected, the wild-type allele morphism analysis, using the polymorphisms identified in hSRBC was also retained. However, immunoblotting with hSRBC-specific coding sequence (data not shown). LOH was detected at a frequency antibody failed to detect the wild-type protein products in three of 46% in 15 informative pairs of lung cancer cell lines compared with hSRBC mutant cell lines tested (Fig. 2E), indicating that the wild-type their matched B lymphoblastoid cell lines (data not shown). In a prior allele was silent. These expression studies suggested that down- separate study, LOH was 44% at D11S1338, a marker near hSRBC,in regulation of expression of hSRBC gene occurs commonly in sporadic lung cancer cell lines (36). breast and lung cancers. Mutational analysis of hSRBC gene was then performed by PCR Down-Regulation of hSRBC Expression in Breast and amplification followed by DNA sequencing using primer pairs cov- Lung Cancer Cell Lines Is Associated with Promoter Region ering the whole coding region and exon/intron junctions on 135 Hypermethylation. Selective down-regulation of tumor suppressor genomic DNAs derived from 35 breast cancer cell lines, 6 ovarian gene expression is often associated with abnormal methylation of cancer cell lines, 60 lung cancer cell lines, 10 microdissected breast CpG dinucleotides in the promoter region (38). We submitted the primary tumors, and 24 microdissceted primary lung tumors. Muta- 7466 bp of genomic DNA containing hSRBC to our software program tions were detected in four cancer cell lines (Table 1). Direct sequenc- PANORAMA, which indicated there were three CpG islands (at nt ing of PCR amplified genomic DNA prepared from the 2008 ovarian 1360–1662, 2816–3082, 3338–5636 of the AF408198 sequence; Ref. cancer cell line revealed a heterozygous 1-bp deletion at nucleotide 39). To investigate the mechanism of hSRBC down-regulation in position 89 (G18 frameshift to 23 stop), resulting in the loss of most tumor cells, we examined the methylation status of CpG dinucleotides of the coding region of hSRBC. The genomic DNA of the NSCLC cell in a 210-bp fragment that was 240 bp upstream of the hSRBC line NCI-H358 contains a heterozygous nonsense mutation at nucle- translation initiation codon. This region contained a CCAAT box otide position 604, changing an arginine (CGA) to a termination motif, a TATA box motif, and two Ap-1 consensus-binding sites (Fig. (TGA) codon at residue 190. The SCLC cell line NCI-H510 harbors 3A). Bisulfite genomic sequencing revealed that the methylation status a heterozygous 21-bp deletion at nucleotide position 177, resulting in of CpG dinucleotides was correlated with their hSRBC expression the removal of seven amino acids; SCLC cell line (NCI-H2081) levels (Fig. 3B). In two cell lines (HTB129 and NCI-H2052) that contains a heterozygous missense mutation (C611G), resulting in an express hSRBC at normal level, eight of nine CpG dinucleotides in A192G amino acid substitution. This A192 is conserved among all of this region examined by bisulfite genomic sequencing were completely unmethylated. In 11 tumor cell lines in which expression of 4 Internet address: http://www.ncbi.nlm.nih.gov/genemap/map. hSRBC was undetectable (HTB131, HTB132, UCI 101, 2008, MCF7, 5 Internet address: http://www-shgc.stanford.edu. NCI-H187, NCI-H378, NCI-H1092, HTB19, NCI-H1299, and NCI- 7945

of hSRBC in primary tumor tissues, the methylation study in primary tumor tissues indicated that 3 (60%) of 5 breast tumors and 11 (79%) of 14 lung tumors underwent hSRBC promoter methylation.

DISCUSSION We have provided evidence suggesting that hSRBC is a candidate TSG at chromosome region 11p15.5-p15.4, predominantly undergo- ing epigenetic inactivation by promoter region methylation. Previ- ously, LOH in this region was found to be associated with several types of adult and childhood tumors. Mutations of the hSRBC gene, including frame shift and truncation mutations, were detected in four cancer cell lines, which suggested that although rare, they do occur. Most importantly, we detected a high frequency (30 of 43) of loss of expression of hSRBC mRNA and protein in breast and lung cancer cell lines, with retained hSRBC expression in normal epithelial cultures from these organs. The loss of hSRBC expression is associated with hSRBC promoter region hypermethylation. This mechanism has been suggested as an alternative way to inactivate TSGs (40). Al- though we did not find hSRBC mutations in the limited numbers of

Fig. 2. The expression of the hSRBC gene is down-regulated in a large fraction of primary tumors that we examined, hSRBC promoter region hyper- breast and lung cancer cell lines, but the gene is expressed in normal human bronchial and methylation was observed in 60% of primary breast tumors and 79% breast epithelial cultures. A, immunoblotting analysis of hSRBC expression in NHBE of primary lung cancers, which suggested that inactivation of hSRBC cells, HMECs, and BRCA1 mutant breast cancer cell line HCC1937 (20 ␮g of protein per lane of total cell lysates). B, Northern blot analysis of hSRBC expression in NHBE and gene function by epigenetic mechanisms is common in breast and normal human lung SAECs using 20 ␮g of total RNA per lane. C, immunoblot analysis lung cancers. In addition, the cancer lines with amino acid sequence of hSRBC expression in breast cancer cell lines (100 ␮g of total cell lysate protein loaded alterations retained the wild-type allele yet failed to express hSRBC per lane). D, immunoblot analysis of hSRBC expression in lung cancer cell lines (100 ␮g of total cell lysate protein loaded per lane). E, immunoblot analysis of hSRBC expression and exhibited promoter region methylation. Thus, promoter region in cancer cell lines (50 ␮g of total cell lysate protein loaded per lane) that contain hSRBC methylation appears to be the dominant mode of inactivation of gene mutations. Although the wild-type allele is present in each of these cell lines, the wild-type hSRBC protein is not expressed. All of the immunoblots were performed on hSRBC in human lung and breast cancers. In addition, among the five proteins fractionated on SDS-PAGE gels using the hSRBC-specific monoclonal antibody polymorphisms detected in the hSRBC coding region, three (C59G, (see “Materials and Methods”). In C, D, and E, 50 ␮g of protein of total cell lysates T509C, and C799T) result in amino acid changes and two (C59G and prepared from HCC1937 (BRCA1 mutant breast cancer cell line that is wild-type for and expresses hSRBC protein) was used as a positive control. The membranes were also C799T) occur at a very low frequency. It is possible that such stripped and reblotted with actin-specific polyclonal antibody (Sigma Chemical Co.) to low-frequency, dramatic amino acid sequence alterations may confer confirm approximately equal protein loading in each lane. interindividual variation in hSRBC functions and may result in different cancer risks. SRBC belongs to a superfamily of proteins that thus far has three H1672), we found that 3 (NCI-H378, NCI-H1299, and NCI-H1672) members: SDPR, SRBC, and PTRF. Mouse SDPR (mSDPR; S67386; were completely methylated and 8 were heavily methylated, with 418 aa) was the first to be cloned in a differential hybridization screen, mCpG density ranging from 3.01 to 4.52 per 100 bp. In two cell lines in which SDPR mRNA levels were found to be induced by serum (HTB130 and NCI-H1437) that have reduced hSRBC expression starvation but not by contact inhibition in NIH3T3 cells (34). Later the level, the CpG dinucleotides in the promoter region were also partially human orthologue of mouse SDPR (hSDPR; AF085481; 425 aa) was methylated, with mCpG densities varying from 2.11 to 2.26 per 100 isolated as a substrate and binding protein of PKC-␣ isoform (PKC-␣) bp. HMECs that express hSRBC (Fig. 2A) were found to have all nine (41). Human SDPR resides on the caveolae of the plasma membrane CpG dinucleotides unmethylated (not shown in Fig 3B). In addition, and may help to recruit PKC-␣ to caveolae. Human SDPR is also the expression of hSRBC in MCF7 cells was partially restored by identical to PS-68 (42), a previously characterized phosphatidylserine- treatment with the histone deacetylase inhibitor TSA, whereas treat- binding protein purified from human platelets (43). The overall amino ment with both demethylation reagent 5Ј-azacytidine and TSA addi- acid identity between human and mouse SDPR is 83%, with 92% tionally induced the expression of hSRBC in MCF7 cells (Fig. 3C). identical in the first 307 amino acids. Rat SRBC (D85435; 263 aa), Finally, we found four lung cancer lines (HCC78, NCI-H1993, isolated as a PKC-␦ substrate and interacting protein, shares several H2052, and H2347) that expressed hSRBC and were also heterozy- similarities with SDPR, including binding phosphatidylserine and the gous for single nucleotide polymorphisms within the gene. We per- regulatory domain of PKC in the absence of Ca2ϩ, and being phos- formed reverse transcription-PCR expression analysis on these and phorylated by PKC in vitro (32). HSRBC described in this paper has found that both alleles were expressed. Thus, at least in these cases, an overall amino acid identity of 81% to the rat orthologue, with the we can rule out imprinting of one of the alleles. first 190 amino acids 91% identical. The third member of the family, We examined the methylation status of the hSRBC promoter region PTRF (AF036249; 392 aa), identified in a yeast two-hybrid screen in primary tumor tissues (Fig. 3D). In five breast tumors examined, with terminator TTF-1, induces dissociation of paused ternary rRNA one was largely unmethylated (case 13 mCpG density, 0.90), one was transcription complexes (35). PTRF is also a very conserved protein. partially methylated (case 67 mCpG density, 1.20), and three were Mouse PTRF shows 72% identity to the chicken orthologue (D82079 heavily methylated (cases 2, 32, and 70 mCpG density, Ͼ3.0). In 14 and D26315). The homology among different family members is

NSCLCs examined, 3 were partially methylated (mCpG density, mostly localized in two regions, homologous region 1 at the NH2 between 1.0 and 3.0), 9 were heavily methylated (mCpG density, terminus (aa 20–139 of hSRBC), which includes the leucine-zipper Ͼ3.0), and 2 were completely methylated. Although the available motif that is completely conserved among three members (Fig. 4A), hSRBC-specific antibody did not allow us to examine the expression and homologous region 2 in the middle (172–194 aa of hSRBC), 7946

Fig. 3. CpG sites in the hSRBC promoter region are methylated in human cancers and correlate with hSRBC protein expression, and hSRBC undergoes expression after treatment with 5Ј-azacytidine and TSA. A, the sequence of the region that was subjected to analysis. Boxed and indicated underneath, 1 CCAAT, 1 TATA, and 2 Ap-1 sites. Bold and underlined, CpG dinucleotides; labeled underneath, the eight CpG dinucleotides that were analyzed for methylation status. Underlined, the primer regions. Numbers (Ϫ451, Ϫ284, Ϫ241) above and below the sequence, the distance to the translation initiation codon. B, the hSRBC expression level is correlated with the methylation status of CpG dinucleotides in the promoter region. The methylation status of each CpG dinucleotide as labeled in A was determined by the sequencing of sodium bisulfite-treated genomic DNA. The treatment converts unmethylated C to T, but leaves methylated C as C. If only T is detected at this position in sequencing, the status of this CpG dinucleotide is defined as fully unmethylated (Ⅺ), which was counted as zero in the calculation of mCpG density. If only C is detected at this position in sequencing, the status of this CpG dinucleotide is defined as fully methylated (f), which was counted as 1 in the calculation of mCpG density. Accordingly, a 50% filled square represents C and T detected at equal peak height in sequencing at this position; thus, the status of this CpG is defined as one-half methylated and one-half unmethylated and was counted as 0.5 in the calculation of mCpG density. A 75% filled square represents that both C and T were detected in sequencing at this position, but the height of C peak was larger than that of T peak; thus the status of this CpG is defined as 75% methylated and 25% unmethylated and was counted as 0.75 in the calculation of mCpG density. A 25% filled square indicates that both C and T were detected in sequencing at this position, but the height of T peak was larger than that of C peak; therefore, the status of this CpG is defined as 75% unmethylated and 25% methylated and was counted as 0.25 in the calculation of mCpG density. The mCpG density per 100 bp was calculated as summing the scores of all of the methylated CpGs from Ϫ451 bp to Ϫ284 bp, divided by 166 bp (from Ϫ451 bp to Ϫ284 bp) and multiplying by 100. ϩϩϩ, normal expression level as in A. ϩ, reduced but detectable expression level; Ϫ, undetectable level by immunoblot. C, treatment of 5Ј-azacytidine and TSA induces hSRBC expression in MCF7 breast cancer cells. MCF7 cells were treated with different concentrations of 5Ј-azacytidine and TSA as indicated above, and the induction of hSRBC expression was examined by immunoblotting with hSRBC-specific monoclonal antibody. D, methylation data of primary tumor samples. The mCpG density was calculated as described above. which contains the putative PKC phosphorylation site (Fig. 4B). has an aspartic/glutamic acid content above 40% (aa 143–173 of Strikingly, in addition to the sequence similarity, the domain organi- hSRBC). Immediately after the acidic region is a basic region (aa zation patterns among the three members are very similar (Fig. 4C). 174–197 of hSRBC) that contains about 38% basic residues and that Each of them has the homologous region 1 that contains the leucine- overlaps and extends the second homologous region. Both SDPR and zipper motif at the NH2 terminus, followed by an acidic region that PTRF have a second acidic region (30% aspartic/glutamic acid) after

Fig. 4. The structural resemblance among SRBC family members. A, alignment of the leucine-zipper-like motif in homologous region 1 among SRBC family members. Filled circles, putative PKC binding sites. C, the schematic representation ,ء .residues involved in the leucine-zippers. B, alignment of homologous region 2 containing the putative PKC binding sites of the domain structures of the three members of SRBC family. Numbers above the bars, amino acid positions. LZ, leucine zipper; HR, homologous region; A, acidic region; B, basic region; PR, Pro-rich region. 7947

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2001 American Association for Cancer Research. INACTIVATION OF hSRBC IN BREAST AND LUNG CANCER the basic region. SRBC differs from SDPR and PTRF in that it has a D11S12 with histology, stage, and metastases in lung cancer. Cancer Detect. Prev., proline-rich region (31% P) instead. The sequence and structural 22: 14–19, 1998. 5. Tran, Y. K., and Newsham, I. F. High-density marker analysis of 11p15.5 in resemblance suggests that the three members may have similar func- non-small cell lung carcinomas reveals allelic deletion of one shared and one distinct tions that are yet to be discovered. region when compared to breast carcinomas. Cancer Res., 56: 2916–2921, 1996. 6. Vandamme, B., Lissens, W., Amfo, K., De Sutter, P., Bourgain, C., Vamos, E., and Although the biochemical function of SRBC is still unknown, De Greve, J. Deletion of chromosome 11p13–11p15.5 sequences in invasive human several lines of evidence suggest that it may have a tumor suppressor ovarian cancer is a subclonal progression factor. Cancer Res., 52: 6646–6652, 1992. function. The expression patterns of SDPR and SRBC under various 7. Viel, A., Giannini, F., Tumiotto, L., Sopracordevole, F., Visentin, M. C., and Boiocchi, M. Chromosomal localisation of two putative 11p oncosuppressor genes growth conditions are very similar (32, 34). The mRNA levels of both involved in human ovarian tumours. Br. J. Cancer., 66: 1030–1036, 1992. SRBC and SDPR are induced on serum starvation and are down- 8. Shaw, M. E., and Knowles, M. A. Deletion mapping of chromosome 11 in carcinoma regulated during G -G transition, which suggests that they may be of the bladder. Genes Chromosomes Cancer, 13: 1–8, 1995. 0 1 9. Baffa, R., Negrini, M., Mandes, B., Rugge, M., Ranzani, G. N., Hirohashi, S., and involved in cell cycle control. 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A common between BRCA1 and hSRBC is currently under active investigation, region of loss of heterozygosity in Wilms’ tumor and embryonal rhabdomyosarcoma but this potential interaction suggests the possibility that hSRBC may distal to the D11S988 locus on chromosome 11p15.5. Hum. Genet., 97: 163–170, 1996. act in the BRCA1 pathway. Mutations in the BRCA1 gene are respon- 15. Visser, M., Sijmons, C., Bras, J., Arceci, R. J., Godfried, M., Valentijn, L. J., Voute, sible for familial breast cases; more than 80% of families with both P. A., and Baas, F. Allelotype of pediatric rhabdomyosarcoma. Oncogene, 15: breast and ovarian cancers carry germ-line BRCA1 mutations, and the 1309–1314, 1997. 16. Albrecht, S., von Schweinitz, D., Waha, A., Kraus, J. A., von Deimling, A., and inheritance of a mutant BRCA1 accounts for 45% of families with Pietsch, T. Loss of maternal alleles on chromosome arm 11p in hepatoblastoma. breast cancer only (48). 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Xie L. Xu, Leeju C. Wu, Fenghe Du, et al.

Cancer Res 2001;61:7943-7949.

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