Frequent inactivation of RASSF1A, BLU, and SEMA3B on 3p21.3 by promoter hypermethylation and allele loss in non-small cell lung cancer

Cancer Letters 225 (2005) 131–139 www.elsevier.com/locate/canlet

Frequent inactivation of RASSF1A, BLU, and SEMA3B on 3p21.3 by promoter hypermethylation and allele loss in non-small cell lung cancer Masao Itoa,b, Genshi Itoa,c, Masashi Kondoc, Mika Uchiyamaa,b, Takayuki Fukuia,b, Shoichi Moria,b, Hiromu Yoshiokab, Yuichi Uedab, Kaoru Shimokatac, Yoshitaka Sekidoa,* a

Department of Clinical Preventive Medicine, Nagoya University School of Medicine, Tsurumai 65, Showa-Ku, Nagoya 466-8560, Japan b Department of Thoracic Surgery, Nagoya University School of Medicine, Nagoya 466-8560, Japan c Department of Respiratory Medicine, Nagoya University School of Medicine, Nagoya 466-8560, Japan Received 19 August 2004; received in revised form 25 October 2004; accepted 28 October 2004

Abstract Non-small cell lung cancer frequently shows loss of heterozygosity of the chromosome 3p21.3 region and several genes such as RASSF1A, BLU, and SEMA3B have been identified as candidate tumor suppressor genes at this region since their downregulation and hypermethylation at their promoter regions were frequently detected in lung cancer. To determine whether these three genes are simultaneously inactivated during lung cancer development, we studied 138 primary non-small cell lung cancers for the promoter methylation status of these genes and allelic loss of the chromosome 3p21.3 region. We found promoter hypermethylation at 32% in RASSF1A, 30% in BLU, and 47% in SEMA3B. Allelic loss of 3p21.3 was detected in 54 (58%) of 93 informative tumors. Despite the weak association of methylation status among these three genes, there was no correlation between the methylation status of each gene and loss of heterozygosity. We also studied possible genes downstream of RASSF1A in 16 primary non-small cell lung cancers and found that the expressions of SM22 and SPARC were significantly downregulated in RASSF1A-hypermethylated tumors. Our results showed that, while candidate tumor suppressor genes at this locus can be simultaneously inactivated by epigenetic alterations, loss of heterozygosity without any hypermethylation of the three genes can also occur in some cases, suggesting that just one allelic loss might also be sufficient for the inactivation of any of these genes for lung cancer development. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Hypermethylation; Non-small cell lung cancer; Tumor suppressor gene; Chromosome 3p21

Abbreviations: LCM, laser capture microdissection; LOH, loss of heterozygosity; MSP, methylation specific PCR; NSCLC, non-small cell lung cancer; PCR, polymerase chain reaction; TSG, tumor suppressor gene. * Corresponding author. Tel.: C81 52 744 1974; fax: C81 52 744 1975. E-mail address: [email protected] (Y. Sekido). 0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.10.041

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1. Introduction Allelic loss of chromosome 3p is a critical early event in the development of lung cancer. Detailed allelotyping analyses have shown that there are several distinct regions on chromosome 3p [1–4], and the 3p21.3 region, one of the most frequently targeted regions, was narrowed by the discovery of overlapping homozygous deletions in three lung cancer cell lines [5]. The 3p21.3 homozygous deletion region was further subdivided by a nesting breast cancer homozygous deletion into two gene sets; eight genes were located within the combined breast and lung cancer 120 kb critical region, and eleven genes were located in the rest of the lung cancer nested homozygous deletions [6]. Extensive analysis for mutations and reduced mRNA levels of these genes compared to normal lung tissues revealed that several genes including RASSF1A, BLU, FUS1, and SEMA3B are frequently downregulated in lung tumors, suggesting that they are the strong candidates for tumor suppressor genes (TSGs) at this locus [7–11]. Furthermore, three of these genes (RASSF1A, BLU, and SEMA3B) have also been shown to be inactivated by tumor-acquired DNA methylation at their promoter regions in lung cancer cells [9,12–14]. Besides lung cancer, frequent inactivation of RASSF1A due to promoter methylation has been shown in a variety of other tumor types, such as brain, breast, ovarian, esophageal, bladder, prostate, nasopharyngeal, and renal cell carcinomas [9,13,15–22]. Recently, RASSF1A has been shown to induce cell cycle arrest by inhibition of cyclin D1 accumulation [23]. Another study showed that RASSF1A regulates the stability of mitotic cyclins and the timing of mitotic progression by inhibiting APC-Cdc20 [24]. Although this evidence suggested that the inactivation of RASSF1A is important in the cell cycle regulation of tumor cells, Agathanggelou et al. identified 66 genes that were inducible by transfection of RASSF1A, including SM22, SPARC, and SDHB, suggesting that RASSF1A may also have multiple functions [25]. Meanwhile, BLU and SEMA3B have been suggested to be TSGs due to the occasional occurrence of missense mutations as well as the loss of their expression by promoter methylation in lung cancer [12,14]. Promoter hypermethylation of BLU was also

detected in 70% of nasopharyngeal carcinomas [26]. BLU contains a predicted MYND domain that is frequently found in some transcription repressors, suggesting that BLU may be involved in important transcriptional regulation pathways. A recent report showed that BLU-transfected neuroblastoma and lung cancer cells resulted in 40–80% inhibition of colony formation efficiency, strongly supporting the notion that BLU is a tumor suppressor [12]. SEMA3B, located in the 250 kb homozygous deletion overlapping region, is a member of the semaphorin family that provides the guidance signals for axons and dendrites [7]. Tomizawa et al. showed that overexpression of SEMA3B induces apoptosis in lung cancer cells, suggesting that it plays an important role in the pathogenesis of lung cancer [27]. A recent study has suggested that SEMA3B is a direct transcriptional target of p53 and may be involved in the p53dependent apoptosis in cancer cells [28]. Thus, the individual status of promoter hypermethylation of RASSF1A, BLU, or SEMA3B has been studied in non-small cell lung cancer, showing that each gene is frequently inactivated in primary lung tumors. However, there have been only a few reports that have simultaneously examined the methylation status of more than two genes at this locus and no report coupled with allelic loss analysis. In this study, to clarify whether simultaneous inactivation of these candidate TSGs is necessary for lung cancer development, we examined the frequencies of hypermethylation of RASSF1A, BLU, and SEMA3B in 138 NSCLCs using a methylation-specific polymerase chain reaction (MSP), analyzed the relationship of methylation status among these three genes, and examined the loss of heterozygosity (LOH) status at 3p21.3 using a microdissection technique. Furthermore, we compared the expression of the possible genes downstream of RASSF1A between methylated and unmethylated primary NSCLCs. We found the promoter hypermethylation of RASSF1A at 32%, BLU at 30%, and SEMA3B at 47%, and the allelic loss of 3p21.3 in 54 (58%) of 93 informative tumors, with inactivation of more than two genes also being identified with or without LOH. These results showed that simultaneous as well as individual inactivation of one or both alleles of these three genes can occur in primary lung cancers. Furthermore, the expression levels of SM22 and SPARC were significantly reduced

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in the RASSF1A-hypermethylated tumors, suggesting that these genes are also regulated by RASSF1A and play some role in the carcinogenesis of primary lung cancers.

2. Materials and methods

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fluorescence-labeled. PCR products amplified from the microdissected tumor specimen DNAs and corresponding noncancerous DNAs were electrophoresed on an ABI 377 and analyzed using GeneScan 3.1 software (PE Applied Biosystems, Foster City, CA). LOH was defined as a more than 40% reduction of intensity as compared with that seen in the corresponding normal control.

2.1. Tissues 2.4. Northern blot analysis Primary lung tumors and corresponding normal tissue were obtained from 138 patients who underwent surgery for NSCLC at the Nagoya University Hospital, Nagoya First Japan Red Cross Hospital, Nagoya Second Japan Red Cross Hospital, Kasugai Municipal Hospital, Chukyo Hospital, and Ogaki Municipal Hospital in Japan. Ethical approval was obtained from each of the six hospitals and fully informed consent from all patients prior to tissue collection. Histological classification was performed according to the third World Health Organization/International Association for the Study of Lung Cancer (WHO/IASLC) [29]. The material comprised 101 adenocarcinomas, 30 squamous cell carcinomas, 2 large cell carcinomas, and 5 adenosquamous cell carcinomas. DNA and RNA were prepared from these samples by the standard technique [30]. 2.2. Methylation analysis Genomic DNAs were modified by bisulfite treatment using CpGenome DNA Modification Kit (Intergen, Purchase, NY) in accordance with the manufacturer’s instructions. Modified DNA was amplified by two different sets of primers specific for unmethylated and methylated gene promoter region sequences. Primer sequences of the promoter regions of RASSF1A, BLU, and SEMA3B were synthesized as previously described [9,12,14]. PCR products were separated in 3% agarose gels and visualized under UV illumination.

Northern blot was performed as previously described [32]. Ten micrograms of total RNAs were electrophoresed in formaldehyde-1.0% agarose gel and transferred to Hybond-NC membrane (Amarsham Pharmacia Biotech, Buckingham, UK). The probes were made by reverse transcription (RT)-PCR. Primer sequences were 5 0 -TCCAGGAGAACTTCCAAGGA-3 0 and 5 0 -TCTCTGTGAATTCCCTCT TA-3 0 for SM22, 5 0 -ACTTCTTTGCCACAAAGTGC-3 0 and 5 0 -GTACTTGTCATTGTCCAGGT-3 0 for SPARC, 5 0 -ACAGCTCCCCGTATCAAGAA-3 0 and 5 0 -GAGTCAATCATCCAGCGATA-3 0 for SDHB, and 5 0 -ATCAAGCCGCACATGCGGAAG-3 0 and 5 0 -GCAAAGGTATAATCTGTAGC-3 0 for CCND3. The membranes were hybridized with a-32P-labeled probes synthesized with random primers and subjected to autoradiography after washing according to the manufacturer’s instructions. The mRNA levels of SM22, SPARC, SDHB, CCND3, and b-actin, were quantified with an Imaging analyzer (BAStation, FUJIFILM, Tokyo, Japan). b-actin was used for normalization. 2.5. Statistical analysis Statistical analysis was performed using Fisher’s exact test, Student’s t test, and the c2-test; P!0.05 was regarded as statistically significant.

3. Results 2.3. LOH analysis Laser capture microdissection (LCM) of tumor specimens was performed as described previously [31]. The microsatellite markers used were D3S1568 and D3S1621, with one of each primer set

3.1. Methylation of RASSF1A, BLU and SEMA3B and clinicopathological status of lung cancer patients We first analyzed the methylation status of the promoter regions of RASSF1A, BLU, and SEMA3B

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Fig. 1. Methylation-specific PCR for the detection of methylated promoters of RASSF1A, BLU and SEMA3B in primary NSCLCs. Case KD178 showed methylation of all three genes, but case 264 showed none of them. T, tumor; N, noncancerous lung tissue; M, results with the primers specific to the methylated sequences; U, results with the primers specific to the unmethylated sequences.

in 138 primary NSCLCs using MSP (Fig. 1). We found that the promoter was methylated in 44 (32%) of RASSF1A, 42 (30%) of BLU, and 65 (47%) of SEMA3B (Table 1). Unmethylated bands

were detected in all primary NSCLC samples. Methylated bands were not detected in the noncancerous lung tissues. To determine whether the methylated status of each gene at the promoter region is associated with the clinical status of the patients, we compared them in terms of age, gender, smoking, histological type, tumor differentiation, and pathological stage (Table 1). However, the methylation status of RASSF1A and BLU did not show any significant correlation with these clinical factors. In contrast, there was a weak, but statistically significant relationship between promoter methylation at SEMA3B and tumor differentiation (PZ0.04). Next, we investigated the possible relationship in methylation status of each set of two genes from the three. Although statistically significant associations of simultaneous methylation status were found between RASSF1A and BLU (PZ0.001), BLU and SEMA3B (PZ0.0004), and RASSF1A and SEMA3B (PZ0.04) in NSCLC, there were also cases with unmatched methylation status of each set (96 matched and 42 unmatched tumors for the RASSF1A and BLU

Table 1 Clinicopathological status and Gene promoter methylation of RASSF1A, BLU, and SEMA3B in 138 NSCLC patients Methylation status RASSF1A

Overall Age Gender Smoking history (pack-yrs) Histological type

Differentiation

p-stage a b c

(Mean, yr) Male Female !20 S20 Adeno Squamous Adenosquamous Large Well Moderate Poorly Stage I/II Stage III/IV

Student’s t test. Fisher’s exact test. c2-test.

BLU

(C)

(K)

44 63.8 29 15 18 26 31 13 0

94 64.4 56 38 47 47 70 17 5

0 10 23 11 35 9

2 34 43 17 63 31

P 0.74a 0.57b 0.36b 0.19c

0.44b

0.16b

SEMA3B

(C)

(K)

42 62.4 28 14 19 23 31 9 2

96 65.0 57 39 46 50 70 21 3

0 13 19 10 30 12

2 31 47 18 68 28

P 0.13a 0.45b 0.85b 0.78c

0.90b

0.99b

(C)

(K)

65 62.6 46 19 26 39 47 14 3

73 65.5 39 34 39 34 54 16 2

1 15 31 19 46 19

1 29 35 9 52 21

P 0.07a 0.06b 0.12b 0.94c

0.04b

0.99b

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microdissection (LCM) using two microsatellite markers, D3S1568 and D3S1621. Among 93 tumors that were informative for at least one of these markers, loss of heterozygosity (LOH) was detected in 54 (58%) and retention of the two alleles was found in 39 (42%) samples (Fig. 2, Table 2). Although there was no statistically significant correlation between LOH and the promoter hypermethylation status of RASSF1A, BLU, or SEMA3B, among the informative 93 cases, 38 (41%) showed both LOH and hypermethylation of at least one of these three genes. Noticeably, among the 54 cases with LOH, 16 did not show the promoter hypermethylation of any of these genes. In contrast, only 11 (12%) of the 93 tumors did not show either methylation of any of three genes or allelic loss (Table 2). 3.3. Downregulation of possible genes downstream of RASSF1A in lung cancer Fig. 2. Microsatellite analysis of the 3p21.3 region. Three representative cases (KD286, KD306, and KD691) showed LOH with both D3S1568 and D3S1612 markers.

To determine whether the expression of candidate genes downstream of RASSF1A is altered by RASSF1A inactivation, we examined four genes including SM22, SPARC, and SDHB, which have been shown to be upregulated due to more than a fivefold change by introduction of RASSF1A into a NSCLC cell line, and CCND3 which has been downregulated by less than a 0.5-fold change [25]. We selected 16 primary NSCLCs including 8 tumors that showed hypermethylation in the RASSF1A promoter and 8 tumors that did not. We found that average levels of SM22 and SPARC mRNA expression in the methylated tumors were significantly lower than those in the unmethylated tumors, with levels of 60 and 50% for SM22 and SPARC, respectively (Fig. 3). Although no statistically

methylation status, 91 matched and 47 unmatched tumors for the BLU and SEMA3B methylation status, and 81 matched and 57 unmatched tumors for the RASSF1A and SEMA3B methylation status), suggesting that methylation can also be induced independently. 3.2. Allelic loss of 3p21.3 in lung cancer To determine how frequently allelic loss of chromosome 3p21.3 occurs with a combination of each gene hypermethylation in NSCLCs, we studied the 138 primary lung cancers with laser capture

Table 2 Gene promoter methylation and allelic loss of 3p21.3 among the 93 informative NSCLC Methylation status RASSF1A (C) LOH (C) (nZ54) (K) (nZ39) a b

20 11

BLU (K) 34 28

P

(C) b

0.50

22 13

alla

SEMA3B (K) 32 26

Promoter hypermethylation found in at least one of three genes. Fisher’s exact test.

P

(C) b

0.52

29 19

(K) 25 20

P b

0.69

(C)

(K)

P

38 28

16 11

0.99b

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Fig. 3. Analysis of expression of candidate RASSF1A downstream genes in lung cancers. (A) Northern blot analysis of SM22, SPARC, SDHB, CCND3, and b-actin in primary NSCLC specimens. SM22 and SPARC mRNA were strongly reduced in the RASSF1A-methylated lung cancers. (B) Expression levels of each gene in the primary tumors were normalized with b-actin and plotted. The levels in normal lung tissues were set at 1.0.

significant differences were found, the expression levels of SDHB and CCND3 between methylated and unmethylated samples showed tendencies as expected: the average level of SDHB expression was higher in the unmethylated samples, and the average level of CCND3 expression was lower in the unmethylated samples (Fig. 3).

4. Discussion In the current study, we investigated the frequencies of the promoter hypermethylation of RASSF1A, BLU, and SEMA3B that are the candidate TSGs located on chromosome 3p21.3 in 138 NSCLCs. The methylation rates were 32% in RASSF1A, 30% in

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BLU, and 47% in SEMA3B. We also examined allelic loss of 3p21.3 and found that 58% of the 93 informative tumors had LOH. The frequencies of hypermethylation in the promoter regions of RASSF1A and SEMA3B were almost consistent with previous studies which reported the frequencies of hypermethylation for RASSF1A and SEMA3B as 30–40% and 40–50%, respectively [9,13,14,33,34]. Meanwhile, we detected hypermethylation of BLU in 30% tumors, which seemed to be slightly higher than a previous study that reported it to be 20% [12]. This slight difference in the frequency of BLU methylation might reflect differences of either background or ethnicity of the patients. Thus, our study coupled with the previous study showed that SEMA3B was more frequently methylated than RASSF1A and BLU, suggesting SEMA3B is one of the most frequently inactivated TSGs in this region. On the other hand, the frequency of LOH at the 3p21.3 region in our study was nearly consistent with previous studies that reported it to be 41–74% [4,14,33]. In addition, Ochi et al. reported that SEMA3B might be one of the mediators involved in the p53-dependent apoptosis pathway [28]. Since we had studied p53 mutations in 115 of the 138 tumors and found that 53 tumors had p53 alterations [35], we compared them with the methylation status of SEMA3B. However, we did not find any correlation between them (data not shown). Despite increasing evidence of the importance of inactivation of candidate TSGs located on 3p21.3 in tumorigenesis, there are several critical questions that remain unclear. For example, is simultaneous inactivation of the genes by promoter hypermethylation necessary for the development of lung cancer? Is there any gene that can be inactivated by allelic loss of 3p21.3, which just reduces its expression by half? In this regard, there have been only a few reports in which the relationship of methylation status of a couple of genes in this region was investigated. For example, in terms of methylation status of RASSF1A and BLU, one study reported no correlation in SCLC, neuroblastoma or glioma, while another indicated a significant association in NSCLC [12,36]. In the current study, we found a weak but significant correlation of the promoter methylation status between the two genes, suggesting that regional hypermethylation at this locus can also be induced in a subset of lung cancers.

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Furthermore, we also compared the relations of the methylation status of each gene and LOH to study in more detail how these genes are inactivated in lung cancer cells. However, we did not observe any relationship between LOH and the methylation status of the three genes, although other groups showed that RASSF1A and SEMA3B methylation status was significantly associated with LOH in 3p21.3 [14,33]. Although there was no direct evidence, it has also been speculated that one of the 3p21.3 TSGs may possibly work as a haploinsufficiency gene [37]. In this regard, our study interestingly showed that 16 cases with LOH had no methylation of any of these three genes, suggesting that at least one allele of these three TSGs was inactivated by just an allelic loss in some lung cancers, although there is the possibility that we missed other inactivation mechanisms such as point mutations. These findings may support the hypothesis that gene inactivation at 3p21.3 due to haploinsufficiency may also be involved in lung cancer development. Among the genes investigated in the current study, RASSF1A has been thought to play an important role in a variety of human carcinomas including NSCLCs, although the function of this gene is still unclear. Agathanggelou et al. identified 66 genes including SM22, SPARC, SDHB, and CCND3 as RASSF1A targets using RASSF1A-transfected cancer cell lines [25]. To determine whether expressions of genes are really altered in primary lung tumors, we studied 16 tumors with 8 RASSF1A-methylated and 8 unmethylated tumors. We demonstrated that the expression levels of SM22 and SPARC were significantly lower in the RASSF1A-methylated tumors than in the unmethylated-tumors, indicating that these two genes are possible RASSF1A targets in primary lung cancers as well as the RASSF1A-transfected cell lines. The importance of SPARC in lung cancer development may also be supported by other studies which report alterations in SPARC expression in a variety of solid tumors, such as breast cancers, colon cancers, esophagus cancers and neuroblastomas [38–42]. Recently, the loss of SPARC expression has been shown to be associated with aberrant hypermethylation of its CpG island in pancreatic adenocarcinomas [43], and SPARC has also been shown to be a multifunctional matricellular protein that modulates cell adhesion and growth [42,44]. Thus, our data

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indicating that SPARC may also play a suppressive role in NSCLC carcinogenesis may require a further functional study in more detail using NSCLC cells. In conclusion, we have shown that primary NSCLCs have frequent methylation of the promoter regions of RASSF1A, BLU, and/or SEMA3B, and that the hypermethylation of one of these genes occurs rather simultaneously with the other gene methylation, with or without LOH at 3p21.3. Furthermore, we demonstrated that the RASSF1A promoter methylation is associated with the downregulation of the candidate downstream genes, SM22 and SPARC, suggesting that RASSF1A also has multifunctional roles in tumorigenesis of NSCLC. Thus, although the inactivation of candidate TSGs of 3p21.3 is a critical event in lung cancer development, rather complicated mechanisms may also underlie the inactivation.

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Acknowledgements This research was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science. We would like to acknowledge Dr Koichi Fujita, Dr Norio Mukoyama, Dr Naohito Sato, and Dr Norio Maeda for the surgical specimens and Ms Hiroko Kako for technical support.

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