Altered Expression of the Fragile Histidine Triad Gene in Primary Gastric Adenocarcinomas

Biochemical and Biophysical Research Communications 284, 850 – 855 (2001) doi:10.1006/bbrc.2001.5038, available online at http://www.idealibrary.com on

Altered Expression of the Fragile Histidine Triad Gene in Primary Gastric Adenocarcinomas Sang-Han Lee,* ,1 Woo-Hyoung Kim,* Hyun-Kyoung Kim,* Kee-Min Woo,* Hae-Seon Nam,† Hong-Soo Kim,‡ Jin-Gook Kim,§ and Man-Hee Cho* *Department of Biochemistry, †Department of Parasitology, ‡Department of Internal Medicine, and §Department of Anatomy, College of Medicine, Soonchunhyang University, Cheon-An 330-090, Korea

Received May 15, 2001

Genomic alterations and abnormal expression of the fragile histidine triad (FHIT) gene in gastric carcinomas were examined to determine whether the FHIT gene is actually a frequent target for alteration during gastric carcinogenesis. To correlate DNA and RNA lesions of the FHIT gene with the effect on FHIT protein expression, we have investigated the FHIT gene for loss of heterozygosity (LOH), aberrant transcripts, point mutations, and protein expression in 35 gastric adenocarcinomas. Allelic loss at D3S1300 was detected in 7 of 33 (21%) informative cases. Aberrant transcripts, with deletions and/or insertions, were observed in 20 of 35 (57.1%) cases and resulted from alternative splicing through exon skipping and/or insertion of the FHIT intron 5 sequence or activation of the cryptic splice site. Point mutations were not found in the FHIT coding region but detected in noncoding exon 2, 3, 4, or 5 of eight aberrant transcripts. Significant reduction of FHIT protein expression was observed in 22 of 35 (62.9%) cases. Aberrant FHIT transcription was shown to be associated with loss of FHIT protein expression. However, aberrant FHIT transcripts themselves were not associated with any clinicopathological parameters, such as age, sex, tumor site, or clinical stage. Moreover, there was no association between the presence of LOH at D3S1300 and the expression of aberrant FHIT transcripts. Nevertheless, high frequency of aberrant FHIT transcripts, significant rate of LOH at D3S1300, and altered expression of the FHIT protein indicate that alterations of the FHIT gene can play an important role in gastric carcinogenesis. © 2001 Academic Press

Abbreviations used: FHIT, fragile histidine triad; LOH, loss of heterozygosity; RT-PCR, reverse transcriptase–polymerase chain reaction. 1 To whom correspondence and reprint requests should be addressed at Department of Biochemistry, College of Medicine, Soonchunhyang University, 366-1, Ssang-Yong Dong, Cheon-An, ChoongNam 330-090, Korea. Fax: 82-41-577-0450. E-mail: L1037624@ sch.ac.kr. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

Gastric cancer is the most common malignancy and the leading cause of cancer deaths in Korea, and is the second most frequent cause of cancer-related death in the world. Although the precise mechanism involved in gastric carcinogenesis remains uncertain at the present time, it is likely that there are certain genetic alterations in gastric epithelial cells that permit them proliferate and metastasize. Recently, it has been suggested that a locus on human chromosome 3p14.2 plays a role during gastric carcinogenesis because of the high frequency of LOH and homozygous deletion in this region (1, 2). The FHIT (fragile histidine triad) gene, a candidate tumor suppressor gene, was isolated from the human chromosome region within 3p14.2 locus (1) spanning the FRA3B fragile site frequently disrupted in various cancers (3). This site has also been reported to contain the t(3;8) break point associated with hereditary renal cell carcinomas (4), pSV2neo integration sites (5), and a spontaneous HPV16 integration site (6). Homozygous deletions, lack or reduced expression of FHIT protein, and alteration of FHIT transcription were frequently observed in several types of primary tumors and cell lines, and a significant proportion of such alterations were shown to correlate with the loss of genomic regions encompassing FHIT exons (1, 7, 8). High frequency of aberrant FHIT transcripts in various cancers is the most prominent finding. However, there has been conflicting data concerning the alterations of the FHIT gene in gastric cancer. A study of Tamura et al. (9) showed that no alterations of FHIT transcripts were observed in 23 gastric carcinomas, including LOH in 2 of 16 (13%) tumors, whereas Baffa et al. (8) demonstrated that 11 of 32 (35%) gastric carcinomas expressed aberrant transcripts and that 20 of 30 (67%) tumors exhibited an absence of FHIT protein expression in immunohistochemical analysis. Capuzzi et al. (10) reported that 27 of 55 (49%) gastric adenocarcinomas were immunohistochemically negative in FHIT protein expression. Focusing on these controversies about FHIT alterations in gastric cancer,

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FIG. 1. Allelic losses at D3S1300 locus detected by nonisotopic analysis of PCR products from tumor (T) and matched normal tissues (N) in gastric adenocarcinomas. Case 5 exhibits only microsatellite instability (MSI). Arrowheads indicate allelic loss in tumor DNA.

we wanted to examine as to whether aberrant FHIT transcription reflects genomic alterations, whether it affects FHIT protein expression, or whether point mutations targeting FHIT were present. In this study, we have studied 35 pairs of primary gastric adenocarcinomas for DNA and RNA alterations, loss of FHIT protein expression, and their correlations among the clinicopathological parameters to explore the role of FHIT in gastric cancer. MATERIALS AND METHODS Patients and specimens. Surgically resected tumors and corresponding normal tissues were obtained from patients with primary gastric cancer at the Soonchunhyang University Cheon-An Hospital (Cheon-An, Korea). Samples were taken immediately after resection and divided into two fragments. One portion was frozen in liquid nitrogen and used for DNA and RNA isolation, and the other was processed for immunochemical analysis. LOH analysis. For the analysis of allelic loss at the FHIT locus, genomic DNA was extracted from 35 gastric adenocarcinomas and adjacent normal tissues. LOH was evaluated using D3S1300 microsatellite marker, which is located within intron 5 of the FHIT gene. The primer sequences were obtained from the Genome database (5⬘-AGCTCACATTCTAGTCAGC CT-3⬘ for upstream primer and 5⬘GCCAATTCCCCAG ATG-3⬘ for downstream primer). The PCR amplification was performed in a volume of 50 ␮l containing 0.4 ␮M of upstream primer and downstream primer, 200 ng of DNA, 1⫻ PCR buffer, 1.5 mM MgCl 2, 200 ␮M dNTPs, 5% DMSO, and 2.5 U of AmpliTaq DNA polymerase (Perkin–Elmer, Norwalk, CT). The PCR consisted of an initial denaturation at 94°C for 3 min and 35 cycles at 94°C for 30 s, 60°C for 45 s, and 72°C for 30 s, followed by 7 min postextension at 72°C. Five microliters of the PCR product was diluted (1:1) with a gel loading buffer (98% formamide, 0.1% xylene cyanol, 0.1% bromophenol blue, and 10 mM EDTA; pH 8.0), resolved in 8% denaturing polyacrylamide gel, and subsequently stained with silver nitrate. RT-PCR and cDNA sequencing. Total RNA was extracted from frozen tissues by the total RNA isolation kit (Ambion Inc., Austin, TX) according to manufacturer’s instruction. Reverse transcription was performed in 20 ␮l final volume containing 1 ␮g RNA, 5 mM MgCl 2, 1 mM of each dNTP, and 2.5 U MuLV reverse transcriptase (Perkin–Elmer). The samples were first denatured for 5 min at 70°C and annealed with oligo d(T) 16 primers at room temperature for 3 min, and then 2.5 U MuLV reverse transcriptase were added. Samples were incubated at 37°C for 60 min. The reaction was then stopped by inactivating the enzyme at 95°C for 5 min. Twenty microliters of cDNA reaction was used for the first PCR amplification in a volume of 100 ␮l containing 0.5 ␮M of primers 5U2 (5⬘-CATCCTGGAAGCTTTGAAGCTCA-3⬘) and 3D2 (5⬘-TCACTGGTTGAAGAATACAGG-3⬘), as published by Ohta et al. (1), 1⫻ PCR buffer, 2 mM MgCl 2, and 1.25 U AmpliTaq DNA polymerase (Perkin–Elmer). The PCR consisted of an initial denaturation at 94°C for 3 min and 25 cycles at 94°C for 30 s, 60°C for 1 min, and 72°C for 1 min,

followed by 7 min postextension at 72°C. Amplified products were diluted 20-fold with TE buffer (10 mM Tris–HCl, 1 mM EDTA), and 1 ␮l was used for the second-round PCR by the nested primers 5U1 (5⬘-TCCGTAGTGCTATCTACATC-3⬘) and 3U1 (5⬘-CATGCTGATT CAGTTCCTCTTGG-3⬘) in the same conditions as the first-round PCR, except for 30 cycles reaction instead of 25 cycles. Selected aberrant transcripts were recovered from ethidium bromide-stained gels and were cloned into a pGEM-T vector system according to manufacturer’s instruction (Promega, Madison, WI). DNA inserts from positive colonies were amplified with T7 and SP6 universal primers and sequenced using an ABI Prism BigDye Terminator Cycle Sequencing kit and an ABI 310 sequencer (Perkin–Elmer). DNA sequences were compared to the GenBank databases utilizing the Blast program available to the web site, http://www. ncbi.nlm.nih.gov. Western blot analysis. Protein extracts from the gastric tumors were subjected to SDS–PAGE on 12% gels, and then electrotransferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The membrane was incubated with 1:1,500 dilution of the with rabbit polyclonal anti-GST-FHIT fusion antibodies (Zymed Laboratories Inc., South San Francisco, CA) for 1 h in Tris-buffered saline (Bio-Rad, Hercules, CA) containing 0.1% bovine serum albumin, followed by the incubation with 1:5000 dilution of horseradish peroxidase-tagged goat anti-rabbit secondary antibody (Santa Cruz Inc., Santa Cruz, CA). The signal was visualized using an Enhanced Chemiluminescence detection kit (Amersham, Cleveland, OH).

RESULTS LOH at D3S1300 was detected in 7 of 33 (21%) gastric carcinomas (Fig. 1). Allelic loss was present in 4 of 20 (20%) tumor specimens that expressed aberrant FHIT transcripts. Two cases showed microsatellite instability (MSI). There was no significant correlation between the presence of LOH at D3S1300 and expression of aberrant FHIT transcripts; however, 6 of 7 tumors with LOH at D3S1300 showed lack or decreased expression of the FHIT protein. To study abnormal FHIT transcription in gastric carcinomas, we reverse transcribed mRNA and amplified the resulting cDNAs from 35 tumors and matched normal stomach tissues. In addition to the normalsized (707-bp) band, small-sized bands, presumably corresponding to altered transcripts, was seen in 20 of the 35 tumors, but also in 13 of adjacent normal stomach tissues (Figs. 2A and 2B). A total of 48 aberrant transcripts were characterized from the 20 tumors, although there was a background of nonspecific bands. Sequence analysis of 48 aberrant transcripts showed only deletions of coding and noncoding exons in 30 cases and both deletion and insertions of intron 5 se-

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FIG. 2. Aberrant transcripts of the FHIT gene detected in gastric adenocarcinomas by reverse transcription–polymerase chain reaction (RT-PCR). (A) Amplification of FHIT transcripts in tumor tissues. Lanes 1–7 correspond to cases 1, 3, 5, 6, 9, 11, and 13. (B) Amplification of FHIT transcripts in tumor (T) and matched normal (N) tissues. The normal-sized 707-bp transcript is indicated by an arrowhead.

quence in 18 cases. Deletion of exon 5– 6 was detected in 9 cases, deletion of exon 4 in 7 cases, deletions of exon 3, exon 3– 4, and exon 4 – 6 in 3 cases, respectively, deletion of exon 4 –5 in 2 cases, and deletions of exon 3– 6, exon 5–7, and exon 5– 8 in 1 case of aberrant transcripts, respectively (Fig. 3). Sequence analysis of 18 aberrant transcripts with insertions from 12 different tumors demonstrated that all of insertions oc-

curred in combination with in-frame deletions and replacing exons. The sizes of the inserts were 59-bp (GenBank Accession No. AF152364, nt 39364 –39306), 81-bp (GenBank Accession No. AF152364, nt 96052– 95972), 103-bp (GenBank Accession No. AF020503, nt 150784 –150682), 112-bp (GenBank Accession No. AF152364, nt 39364 –39253), 138-bp (GenBank Accession No. AF152365, nt 39082–38945), 158-bp (GenBank Accession No. AF020503, nt 61914 – 61869 and GenBank Accession No. AF152364, nt 39364 –39253), and 250-bp (GenBank Accession No. AF152365, nt 39082–38945 and GenBank Accession No. AF152364, nt 39364 –39253). Some inserts (59, 81, and 138 bp) were found between various exons (E3, E4, or E5) and the last 69 nt of exon 6. One hundred and three- and 112-bp inserts were the parts of intron 5 sequence flanking on the 3⬘-side of the coding exon 5 and on the 5⬘-side of coding exon 6, respectively. Some inserts were overlapped; a 158-bp comprised the 112-bp and the other 46-bp sequence. The 250-bp insert comprised the 138- and 112-bp insert, respectively. Various patterns of FHIT gene alterations observed in gastric adenocarcinomas with aberrant FHIT transcripts are summarized in Table 1. Table 2 shows point mutations detected in FHIT gene. A3 G mutation was detected in four cases (nt ⫺108 in exon 4 of aberrant transcript 11A, nt ⫺188 in exon 2 of aberrant transcript 16B, and nt ⫺191 in exon 2 of aberrant transcripts 26B and 26C). C3 T mutation was detected in 2 cases (nt ⫺59 in exon 4 of aberrant transcript 18B and nt ⫺82 in exon 4 of aberrant transcript 32A). T3 C mutation was detected in 2 cases (nt ⫺158 in exon 3 of aberrant transcript 21C and nt ⫺14 in exon 5 of aberrant transcript 34B). Western blot analysis of 35 gastric adenocarcinomas and matched normal adjacent tissues showed significant expression of FHIT protein in 22 specimens (Fig.

FIG. 3. A schematic representation of aberrant FHIT transcripts found in gastric adenocarcinomas. The top line shows the intact FHIT cDNA map. The thick and thin bars show the coding and untranslated regions, respectively. The positions of splice sites are shown by downward arrows, according to the nucleotide numbers. Gaps indicate the deleted regions within the FHIT cDNAs. ‚, deletion. 852

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1

Summary of FHIT Gene Alterations in Gastric Adenocarcinomas Insertion Case No.

Age

Sex

Tumor site

TNM stage

AT

Deletion a

1

31

M

Antrum

T 4N 3M 0

3

61

F

Body

T 4N 3M 0

A B C A

5

53

F

Antrum

T 3N 1M 0

B A

6

64

M

Antrum

T 3N 1M 0

9 11

75 70

F M

Antrum Cardia

T 2N 0M 0 T 2N 0M 0

13 14

55 71

M F

Antrum Antrum

T 3N 1M 0 T 4N 2M 0

16

52

M

Antrum

T 4N 3M 0

E3, E4 E5, E6* E5, E6, E7 E5, E6* E5, E6* E10* E4 E4, E5, E6* E10* E5, E6* E4, E5 E4, E5, E6* E3, E4, E5, E6* E5, E6* E4, E5 E10* E5, E6* E5 E5, E6* E3 E5, E6* E5, E6* E5, E6* E5, E6* E4 E3, E4 E3, E4, E5, E6* E5, E6* E5, E6* E5, E6* E5, E6* E4, E5, E6* E5 E5 E5, E6* E5, E6* E4, E5 E4, E5, E6* E4, E5, E6* E3 E4 E3, E4 E4 E6, E7, E8 E5, E6, E7, E8 E4 E4 E3, E4, E5, E6* E4, E5, E6* E3 E4

18

64

F

Cardia

T 3N 1M 0

20

52

M

Antrum

T 3N 2M 0

22

69

F

Antrum

T 3N 3M 0

23 24

40 52

F M

Antrum Antrum

T 3N 1M 0 T 2N 0M 0

26

61

M

Fundus

T 4N 1M 0

28

54

M

Fundus

T 3N 3M 0

30

68

M

Cardia

T 3N 2M 0

32 33

34

62 79

57

M F

M

Antrum Body

Body

T 2N 1M 0 T 1N 1M 0

T 3N 1M 0

B A B C A A A A B C A B C A B A B C A B C D A A B C D A B C A B C A B C A A B C A B

Size (bp)

Homology

⫹138 ⫹112

Intron 5 Intron 5

⫹138

Intron 5

⫹138 ⫹138

Intron 5 Intron 5

⫹138

Intron 5

⫹138

Intron 5

LOH

FHIT protein expression

⫹

Down

⫺

Down

MSI

Down

⫺

⫹

⫺ ⫺

⫹ ⫹

⫺ ⫺

Down Down

⫺

Down

⫺

Down

⫺

⫹

⫹138 ⫹81 ⫹59

Intron 5 Intron 5 Intron 5

⫺

Down

⫹138 ⫹250 ⫹158 ⫹138

Intron Intron Intron Intron

5 5 5 5

⫺ ⫹

Down Down

⫹112 ⫹59

Intron 5 Intron 5

⫹

Down

⫺

Down

⫺

Down

⫹ ⫺

⫹ Down

⫺

Down

⫹103

⫹138

Intron 5

Intron 5

a Exons were not completely deleted; E6* and E10* mean deletion of the first 77 nt within exon 6 and the first 11 nt of exon 10, respectively. AT and MSI mean aberrant transcripts and microsatellite instability, respectively.

4). In 15 of 20 (75%) tumors with aberrant FHIT transcripts, reduction of FHIT protein expression was shown, although a 707-bp normal-sized FHIT tran-

script was expressed (Table 1). Only 7 of 15 (47%) tumors without aberrant FHIT transcripts showed reduced FHIT protein expression.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 2

FHIT Mutation of Gastric Adenocarcinomas Case

Nucleotide number

11A 16B 18B 21C 26B 26C 32A 34B

⫺108 ⫺188 ⫺59 ⫺158 ⫺191 ⫺191 ⫺82 ⫺14

Localization Exon Exon Exon Exon Exon Exon Exon Exon

4 2 4 3 2 2 4 5

Base change A3G A3G C3T T3C A3G A3G C3T T3C

DISCUSSION Early studies suggested that deletions within the FHIT gene might give rise to aberrant FHIT transcripts, resulting in loss of FHIT protein in various tumors (11, 12). To study that these aberrant transcripts could actually be attributed to alterations of the FHIT gene, we performed LOH analysis using the microsatellite marker D3S1300 located within intron 5 of the FHIT gene. The results showed that allelic loss was present in 21% (7/33) of gastric carcinomas. The frequency of allelic loss at D3S1300 ranged from 9.1% (5/55) in a report by Noguchi et al. (13) to 27.6% (8/29) in a report by Gemma et al. (14). The reason for this discrepancy is not clear, but may attribute to tumor specimens derived from very distinct geographical areas. LOH at D3S1300 was found with equivalent frequency between tumors with and without aberrant transcripts. This result suggests that aberrant transcripts found in this study are likely associated with reduced splicing fidelity rather than allelic loss of the FHIT gene. The finding that most of the cases with aberrant transcripts showed more than two aberrant bands supports this possibility. LOH in FRA3B site and chromosomal breakage mostly occur within FHIT intron 5 and, less commonly, intron 4. Here, we have shown that 71% (35/48) of aberrant transcripts with deletion and/or insertion involved exon 5 and/or intron 5 sequence, respectively. It is possible that these aberrations due to the characteristics of the FHIT gene locus as fragile site. Although there may not be a direct relation between DNA alterations within the FHIT gene and occurrence of aberrant transcripts, partial deletions of intronic sequence within the FHIT gene might affect transcription fidelity and thus result in the generation of numerous aberrant transcripts. Most of tumor specimens were shown to have multiple bands. Sequence analysis confirmed that these small-sized bands were transcripts in low abundance that were alternatively spliced. These were derived from the results of exon skipping, and/or insertion of FHIT intron 5 sequence, or selection of cryptic splice site within intron 5 and exon 6 of the FHIT gene. AG

nucleotide at position 179 and 180 of the FHIT cDNA sequence (1) was frequently observed as cryptic splice acceptor site. Also, these aberrant transcripts were non-expressive splicing variants of low abundance because most of the deletions abrogated the exon 5, which contains the translation initiation codon. In this study, reduced expression of FHIT protein was frequently observed in tumor specimens with aberrant FHIT transcripts. This result is consistent with previous report that FHIT protein expression is markedly reduced or absent in primary cervical tumors in which aberrant or absent FHIT transcripts are detected by RT-PCR (15). However, FHIT protein expression was often repressed even in tumors without detectable FHIT mRNA aberration in this study. In this study, 62.9% (22/35) of the cases showed reduced expression of FHIT protein, despite the presence of wild-type FHIT transcript, suggesting that mechanisms other than homozygous exon loss may be related to the defect in FHIT protein expression. Previous reports demonstrated that homozygous deletions in FHIT gene mostly involved intronic sequences within the FHIT gene (4, 16). Alterations within FHIT introns can affect protein expression, possibly through the effect on mRNA stability or efficiency of processing. The FHIT gene has huge intronic sequence, especially introns 4 and 5, which could be the potential location of the actual target gene in various cancers while the FHIT gene is an innocent bystander in the most common fragile site of the human genome, FRA3B. It is also possible that reduced splicing fidelity, represented by exon and/or intron alteration, can produce abnormal gene products, potentially acting as transcription factors, oncogenes, or inhibitors of tumor suppressors. Alternatively, as yet unidentified, this may reflect the possibility of methylation in 5⬘CpG island (17). Furthermore, point mutations were not detected within the coding region of FHIT transcripts. This is consistent with findings from other groups (14, 18). Eight point mutations were detected in noncoding exon 2, 3, 4, or 5 within a variety of aberrant transcripts but their significance need further study. The finding that both aberrant transcripts with and without point mutation have been simultaneously found in one tumor specimen indicates the heterogeneity of tumor cells. No correlations between aberrant FHIT transcripts and age, sex, clinical stage, or tumor site were evident.

FIG. 4. Representative immunoblot analysis of FHIT protein expression in gastric adenocarcinomas (T) and matched normal adjacent tissues (N).

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This finding is consistent with previous results that no difference in clinicopathological factors was found between the groups with and without abnormal transcripts (19). However, reduced expression of FHIT protein was frequently observed in advanced stage (III– IV) of gastric carcinomas (data not shown). This result supports the recent study showing that the absence of FHIT protein correlated with higher tumor stage in gastric adenocarcinomas (10). Also, most of tumors with LOH at D3S1300 showed reduced expression of the FHIT protein. However, none of these associations were found to be statistically significant. In this study, the presence of wild-type transcript and lack of splice site mutation or point mutations in coding region raise questions about the role of the FHIT gene as a classic tumor suppressor gene in gastric tissue. However, we have shown that the FHIT gene is frequently altered in gastric cancer, demonstrating LOH, high frequency of aberrant transcripts, and reduced expression of FHIT protein. Additionally, aberrant FHIT transcripts are associated with loss of FHIT protein expression. Our results indicate that alterations of the FHIT gene can play an important role in gastric carcinogenesis. ACKNOWLEDGMENT This study was supported by a grant from the research promotion fund (20000072) of Soonchunhyang University in 2000.

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