Oncogene (2004) 23, 7095–7103 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc AKAP12/Gravin is inactivated by epigenetic mechanism in human gastric carcinoma and shows growth suppressor activity Moon-Chang Choi1, Hyun-Soon Jong*,1, Tai Young Kim1, Sang-Hyun Song1, Dong Soon Lee2, Jung Weon Lee1, Tae-You Kim1,3, Noe Kyeong Kim3 and Yung-Jue Bang*,1,3 1National Research Laboratory for Cancer Epigenetics, Cancer Research Institute, Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799, Korea; 2Department of Clinical Pathology, Seoul National University College of Medicine, Seoul 110-744, Korea; 3Department of Internal Medicine, Seoul National University College of Medicine, Seoul 110-744, Korea AKAP12/Gravin, one of the A-kinase anchoring proteins Introduction (AKAPs), functions as a kinase scaffold protein and as a dynamic regulator of the b2-adrenergic receptor complex. AKAP12/Gravin, one of the A-kinase anchoring However, the biological role of AKAP12 in cancer proteins (Dell’Acqua and Scott, 1997; Nauert et al., development is not well understood. The AKAP12 gene 1997; Diviani and Scott, 2001; Feliciello et al., 2001), encodes two major isoforms of 305 and 287 kDa was first isolated as a protein recognized by serum (designated AKAP12A and AKAP12B, respectively, in from myasthenia gravis patients (Gordon et al., 1992). this report). We found that these two isoforms are AKAP12 organizes the complex of PKA and PKC independently expressed and that they are probably under (Nauert et al., 1997), and is an important regulator the control of two different promoters. Moreover, both of the b2-adrenergic receptor complex (Shih et al., isoforms were absent from the majority of human gastric 1999; Lin et al., 2000a). AKAP12 expression can cancer cells. The results from methylation-specific PCR be induced by several drugs like phorbol ester (MSP)and bisulfite sequencing revealed that the 5 0 CpG (Gordon et al., 1992; Nauert et al., 1997) and lysopho- islands of both AKAP12A and AKAP12B are frequently sphatidylcholine (Sato et al., 1998), which suggests the hypermethylated in gastric cancer cells. Treatment with participation of AKAP12 in diverse signal transduction DNA methyltransferase inhibitor and/or histone deacety- cascades. lase inhibitor efficiently restored the expression of AKAP12 has been mapped to chromosome 6q24– AKAP12 isoforms, confirming that DNA methylation 25.2, which frequently contains deletions in tumors, is directly involved in the transcriptional silencing of including melanoma (Millikin et al., 1991) and breast AKAP12 in gastric cancer cells. Hypermethylation of cancer (Tibiletti et al., 2000). Interestingly, the down- AKAP12A CpG island was also detected in 56% (10 regulation of AKAP12 expression has been reported in of 18)of primary gastric tumors. The restoration of human prostate cancers in vivo (Xia et al., 2001), AKAP12A in AKAP12-nonexpressing cells reduced suggesting that the inactivation of AKAP12 expression colony formation and induced apoptotic cell death. In may be linked to oncogenesis. Thus, the molecular conclusion, our results suggest that AKAP12A may mechanism of the tumor-specific inactivation of function as an important negative regulator of the survival AKAP12 expression should be defined. pathway in human gastric cancer. The patterns of DNA methylation and chromatin Oncogene (2004) 23, 7095–7103. doi:10.1038/sj.onc.1207932 structure are profoundly altered in neoplasia. Aberrant Published online 19 July 2004 methylation of the CpG islands located in the promoter regions of tumor suppressor genes (TSGs) is now firmly Keywords: AKAP12; alternative promoter; DNA established as a major epigenetic mechanism of gene methylation; histone deacetylation; apoptosis inactivation in tumorigenesis (Jones and Laird, 1999; Esteller, 2002; Jones and Baylin, 2002). Genes silenced by DNA methylation can be restored by treatment with 5-aza-20-deoxycytidine (5-Aza-dC), an inhibitor of DNA methyltransferase (Jones and Taylor, 1980). The src-suppressed C-kinase substrate (SSeCKS), the rodent orthologue of human AKAP12, was originally *Correspondence: Y-J Bang, Department of Internal Medicine, Seoul identified as a gene downregulated in response to Src National University College of Medicine, 28 Yongon-dong, Chongro-gu, and Ras activation (Lin et al., 1995). The overexpression Seoul 110-744, Korea; of SSeCKS suppressed Src-induced oncogenesis by E-mail: [email protected]; H-S Jong, Cancer Research Institute, inhibiting the cellular proliferation, and by reducing Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799, Korea; E-mail: [email protected] anchorage-independent growth in soft agar and inva- Received 16 November 2003; revised 1 April 2004; accepted 6 May 2004; siveness in Matrigel (Lin and Gelman, 1997). A recent published online 19 July 2004 report showed that SSeCKS regulates blood–brain Epigenetic inactivation of AKAP12 M-C Choi et al 7096 barrier differentiation by inhibiting angiogenesis by and SNU-5 cells expressed three isoforms (305, 287 and reducing VEGF expression, and that the constitutive 250kDa). Two isoforms were detected in SNU-484 (305 expression of SSeCKS may be important for brain and 250kDa) and SNU-638 (287 and 250kDa). homeostasis (Lee et al., 2003). Figure 2a is a schematic diagram of the AKAP12 gene In this study, we identified AKAP12 as a novel structure based on Genbank database (Accession no. epigenetic target gene in gastric cancer, and compara- NT_023451). AKAP12A is composed of four exons (1a, tively evaluated the silencing of the two transcripts of 2, 3 and 4), whereas AKAP12B contains three (1b, 3 and AKAP12. We also examined the tumor suppressor 4). To distinguish between the transcripts of AKAP12A activity of AKAP12A in gastric cancer cells. and AKAP12B, we performed 50-RACE using primers derived from exon 3, which is common to the two transcripts. All RACE products using the mRNA from Results SNU-638 encoded exon 1b, but not exon 1a (data not shown), indicating that SNU-638 cells express only Loss of AKAP12 expression in gastric cancer cell line AKAP12B. Notably, the upstream of exon 1b was not detected by 50-RACE, suggesting the existence of an Northern and Western analyses were performed to internal promoter located at the 50 boundary of the examine the expression of AKAP12 in human gastric coding exon 1b. To characterize the expression profiles cancer cells. HepG2 and three gastric cancer cell lines (SNU-5, -484 and -638) were found to express AKAP12 mRNA and protein, whereas the other cells did not (Figure 1). To determine whether AKAP12 inactivation is due to chromosomal deletion, the genomic DNA at the AKAP12 locus was examined by Southern blot and FISH analysis in gastric cancer cells; however, no genetic defects were detected (data not shown). These cell lines provided a panel of AKAP12-expressing and -nonexpressing cells, and were used to further investi- gate the mechanism underlying the loss of AKAP12 expression. Identification of distinct transcripts of AKAP12 The two transcripts of AKAP12 encode three isoforms of 305, 287 and 250 kDa (Xia et al., 2001; Gelman, 2002). One transcript variant 1 (designated AKAP12A in this study) encodes the large isoform (305 kDa), and another transcript variant 2 (designated AKAP12B in this study) has the small isoform (287 kDa), whereas the Mr 250kDa isoform is a proteolytic cleavage product of these two major isoforms. As shown in Figure 1, HepG2 Figure 2 Identification of differentially expressed AKAP12 Figure 1 Expression of AKAP12A mRNA and protein in gastric transcripts. (a) Schematic diagram of the intron/exon structure of cancer cells. Northern blot of total RNA was hybridized with the AKAP12 and splicing patterns. This representation is based on the probe for exon 3 (common to the two AKAP12 transcripts) and human genome sequence contig NT_023451. (b) Northern blots of subsequently with the b-actin probe as a loading control. The total RNA from HEK293, HepG2, SNU-601 (À), SNU-601 expression of AKAP12 protein was determined by Western blot exposed to 10 mM 5-Aza-dC ( þ ), and SNU-638 cells were analysis. A Western blot with anti-Tubulin antibody was used to hybridized with probes for exon 1a´ and exon 2 (P1 and P2), exon control for protein content 1b (P3), exon 3 (P4), and b-actin Oncogene Epigenetic inactivation of AKAP12 M-C Choi et al 7097 of the AKAP12 transcripts by Northern analysis, we complete or partial methylation in eight of 11 gastric designed four kinds of probes specific for each cancer cell lines. In contrast, three cell lines (SNU-5, transcript. Probes, P1 and P2, derived from exon 1a -484 and -668) were unmethylated at the AKAP12A and exon 2 were specific for the AKAP12A transcript. CpG island. In the case of AKAP12B, four cell lines P3 derived from exon 1b was specific for the AKAP12B (SNU-5, -16, -638 and -668) were unmethylated at the transcript. P4 derived from exon 3 was common to the AKAP12B CpG island, whereas the remaining seven cell both transcripts. Figure 2b shows that HepG2 expressed lines were either completely or partially methylated. A both AKAP12 transcripts, whereas HEK293 and close correlation was observed between the expression SNU-638 expressed AKAP12A and AKAP12B, respec- and methylation status of each CpG island, except in tively. These results imply that both AKAP12 isoforms SNU-16 and -668 cells (Figures 1 and 3c). were missing in the majority of gastric cancer cells. Restoration of AKAP12 by 5-Aza-dC treatment Aberrant promoter methylation of AKAP12A and AKAP12B in gastric cancer cells We examined whether demethylation could restore AKAP12 expression in gastric cancer cells. In During the genetic mapping of AKAP12, we found that many cases, treatment with the DNA methyltransferase AKAP12A and AKAP12B have CpG islands in their inhibitor 5-Aza-dC in AKAP12-nonexpressing cells promoter regions.