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MYO18B, a Candidate Tumor Suppressor Gene at Chromosome 22Q12.1, Deleted, Mutated, and Methylated in Human Lung Cancer

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MYO18B, a Candidate Tumor Suppressor Gene at Chromosome 22Q12.1, Deleted, Mutated, and Methylated in Human Lung Cancer MYO18B, a candidate tumor suppressor gene at chromosome 22q12.1, deleted, mutated, and methylated in human lung cancer Michiho Nishioka*†, Takashi Kohno*, Masachika Tani*, Nozomu Yanaihara*, Yoshio Tomizawa*, Ayaka Otsuka*, Shigeru Sasaki*, Keiko Kobayashi*, Toshiro Niki*, Arafumi Maeshima*, Yoshitaka Sekido‡, John D. Minna§, Saburo Sone†, and Jun Yokota*¶ *National Cancer Center Research Institute, Tokyo 104-0045, Japan; †Tokushima University School of Medicine, Tokushima 770-8503, Japan; ‡Nagoya University School of Medicine, Nagoya 466-8550, Japan; and §Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390-8593 Communicated by Webster K. Cavenee, University of California at San Diego, La Jolla, CA, July 26, 2002 (received for review February 27, 2002) Loss of heterozygosity on chromosome 22q has been detected in by the chr 22 sequencing project (11). It was recently proposed approximately 60% of advanced nonsmall cell lung carcinoma that the bk125H2.1 gene be named MYO18B based on the partial (NSCLC) as well as small cell lung carcinoma (SCLC), suggesting the amino acid sequence deduced from the genomic sequence data presence of a tumor suppressor gene on 22q that is involved in lung (12). Thus, we use the name MYO18B for the bk125H2.1 gene in cancer progression. Here, we isolated a myosin family gene, this article. MYO18B, located at chromosome 22q12.1 and found that it is Here we isolated the full-length cDNA, determined the frequently deleted, mutated, and hypermethylated in lung cancers. genomic structure of the MYO18B gene, and analyzed it for Somatic MYO18B mutations were detected in 19% (14͞75) of lung deletion, mutation, expression, and methylation in a large num- cancer cell lines and 13% (6͞46) of primary lung cancers of both ber of lung cancers. The MYO18B gene was frequently altered by SCLC and NSCLC types. MYO18B expression was reduced in 88% several molecular mechanisms, including homozygous͞ (30͞34) of NSCLC and 47% (8͞17) of SCLC cell lines. Its expression hemizygous deletions, intragenic mutations, and hypermethyl- GENETICS was restored by treatment with 5-aza-2؅-deoxycytidine in 11 of 14 ation of the CpG island. Restoration of MYO18B expression in cell lines with reduced MYO18B expression, and the promoter CpG lung cancer cells suppressed colony formation in soft agar. Thus, island of the MYO18B gene was methylated in 17% (8͞47) of lung it was strongly suggested that the MYO18B gene is a target TSG cancer cell lines and 35% (14͞40) of primary lung cancers. Further- of 22q LOH and is involved in the progression of lung cancer. more, restoration of MYO18B expression in lung carcinoma cells suppressed anchorage-independent growth. These results indicate Materials and Methods that the MYO18B gene is a strong candidate for a novel tumor Samples. Seventy-six lung cancer cell lines, consisting of 26 suppressor gene whose inactivation is involved in lung cancer SCLCs and 50 NSCLCs, were used in this study (13). In 10 progression. SCLCs and 15 NSCLCs, corresponding lymphoblast cells were available (14) (details are available on request). Forty-six sur- llelic losses on chromosome (chr) 22q have been frequently gical specimens (16 SCLCs and 30 NSCLCs) and adjacent Adetected in human lung cancers by several methods, such as noncancerous tissues were obtained at surgery. DNA was pre- loss of heterozygosity (LOH) analysis (1–3), cytogenetic analysis pared as described (10). Allelic imbalance (AI) at two micro- (4, 5), and comparative genomic hybridization (6), indicating the satellite loci, D22S429 and D22S538,intheMYO18B locus (Fig. presence of a tumor suppressor gene (TSG) on chr 22q, which is 1A) was examined in these 46 cases by the method described (1). frequently inactivated in lung cancer. We previously reported Forty cases were informative for either or both of the loci, and that the incidence of LOH on 22q in advanced-stage non-small AI was detected in 25 (63%) of the cases. Noncancerous tissue cell lung carcinoma (NSCLC) (Ϸ60%) was significantly higher DNA of 50 unrelated individuals was also used in this study. than that in early-stage NSCLC (Ϸ30%) (1, 2). The incidence of Randomly primed cDNAs for lung cancer cell lines, SAEC LOH on chr 22q was also high in small cell lung carcinomas (Clonetics, Walkersville, MD), NHBE (Clonetics), WI-38, seven (SCLCs) (Ϸ60%) irrespective of clinical stages (3). Thus, it is NSCLCs, and human normal tissues were prepared as described likely that inactivation of a TSG on chr 22q plays an important (10). Alveolar type II cells and bronchiolar epithelial cells were role in the progression of both SCLC and NSCLC types. microdissected by using the LM200 LCM System (Arcturus Three known TSGs, SNF5͞INI1, NF2, and EP300, have been Engineering, Mountain View, CA), and total RNA was prepared mapped to 22q11.2, 22q12.2, and 22q13, respectively. However, with a Micro RNA Isolation kit (Stratagene) (15). Randomly genetic and͞or epigenetic alterations of these genes are infre- primed cDNA was reverse-transcribed from total RNA by using quent in both SCLC and NSCLC (7–9). Therefore, a target SensiScript reverse transcriptase (Qiagen, Tokyo). gene(s) for 22q LOH in human lung cancer is still unknown. Recently, we identified a homozygous deletion on chr 22q12.1 in Isolation of Full-Length MYO18B cDNA. Genome DNA sequences a SCLC cell line, Lu24 (10). The deleted region included the covering the Lu24 deletion region (GenBank accession nos. SEZ6L gene, whose structure had been partially characterized by AL022329, AL080245, Z98949, AL079300, AL022337, the chr 22 sequencing project (11). Therefore, we determined the AL080273, and AL023513) were used to identify exons by the genomic structure of SEZ6L and performed a mutational anal- GENSCAN (16) and BLAST programs (17) after elimination of ysis of this gene in lung cancers. However, SEZ6L was genetically Ͻ altered only in a small subset ( 10%) of lung cancers. Therefore, Abbreviations: LOH, loss of heterozygosity; TSG, tumor suppressor gene; chr, chromosome; it was strongly suggested that another unknown gene(s) on chr SCLC, small cell lung carcinoma; NSCLC, non-small cell lung carcinoma; 5-aza-dC, 5-aza-2Ј- 22q functioned as a major TSG in lung cancer progression. deoxycytidine; TSA, trichostatin A; RTQ-PCR, real-time quantitative PCR. Subsequent analyses of the genomic sequence covering the Lu24 Data deposition: The sequence reported in this paper has been deposited in the GenBank deletion identified a novel myosin heavy chain-like gene, database (accession no. AB075376). bk125H2.1, whose structure also had been partially determined ¶To whom reprint requests should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.192445899 PNAS ͉ September 17, 2002 ͉ vol. 99 ͉ no. 19 ͉ 12269–12274 Downloaded by guest on September 27, 2021 Fragment Analysis and WAVEMAKER software 4.0 (Trans- genomic, Omaha, NE). PCR products with different mobilities in either SSCP or WAVE analysis were purified and directly sequenced. (Primers and conditions are available on request.) -5-Aza-2؅-deoxycytidine (5-aza-dC) and͞or Trichostatin A (TSA) Treat ment. For the first 48 h, cells were incubated with medium containing 1.0 ␮M 5-aza-dC (Sigma), and then for another 24 h with the addition of 1.0 ␮M TSA (Wako, Tokyo). Total RNA was then isolated with a RNeasy minikit (Qiagen), RTQ-PCR was performed as described above, and a relative expression of Ͼ2 compared with basal levels was considered as being induced. Promoter Methylation Analysis. Genomic DNA was treated with sodium bisulfite as described (19). PCR was performed by mixing DNA (Ͻ200 ng) with 50 pM of the CpG-F (5Ј- AAGGTATGTTTATATGTATT-3Ј) and CpG-R (5Ј-CAG- GAAACAGCTACGACAACAAACAAAAAAATCAAAC- 3Ј) primers (Fig. 5, which is published as supporting information on the PNAS web site, www.pnas.org) in a reaction mixture (40 ␮l) containing dNTPs (200 ␮M each) and 1 unit of HotstarTaq DNA polymerase (Qiagen) at 95°C (1 min), 50°C (1 min), and 72°C (4 min) for 40 cycles. The CpG-R primer contained the M13-RV sequence (underlined) as a site to initiate sequencing. PCR products were directly sequenced to obtain average meth- ylation levels. PCR products of five cell lines and two normal lung tissues were subcloned into the pGEM T-Easy vector (Promega) and sequenced. Fig. 1. Structures of the MYO18B gene and MYO18B protein. (A) Physical Definition of Methylation. and transcriptional maps of the region containing the MYO18B gene. STS Plasmid DNA with or without con- markers are marked on the top. Locations of CpG islands and three genes are verted cytosines at CpG sites 10 and 11 was amplified by the SP6 indicated. The region of homozygous deletion in a SCLC cell line, Lu24, is and T7 primers. Then, seven artificial heterozygote samples were indicated by a dashed horizontal line. Exon-intron organization of the prepared by mixing each of the PCR products in an equal ratio MYO18B gene is depicted as vertical bars. Predicted transcriptional start and and sequenced by using the M13-RV primer. The percentage of stop sites are indicated by vertical bars under exons 2 and 43, respectively. methylation was calculated by the formula of the peak height of (B) Schematic diagram of a domain structure of MYO18B protein and positions G divided by the sum of peak heights G and A. The mean values of mutations in lung cancer cells. Numbers on the top indicate codon numbers. of the seven samples were 50.5% at both CpG sites 10 and 11, Closed arrowheads, somatic mutations; open arrowheads, base substitutions and small differences in the percentage of methylation were detected only in cell lines but not in 50 unrelated individuals. Putative func- tional domains are indicated at the bottom. observed because of artificial signal variations (SD at sites 10 and 11 was 2.0% and 1.9%, respectively).
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