[CANCER RESEARCH 52, 6696-6698, December I, 1992]

Advances in Brief

Frequent Somatic Mutations of the APC Gene in Human Pancreatic Cancer 1

Akira Hori i , Shuichi Nakatsuru, Yasuo Miyosh i , Sh ige tosh i lchi i , Hiroki Nagase , Hirosh i Ando, Akio Yanagisawa, Eiju Tsuchiya , Yo Kato, and Yusuke Nakamura 2

Departments of Biochemistry [.4. H., S. N., Y. M., S. I., H. N., 11. A., Y. N.] and Pathology [.4. Y., E. T., Y. K.], Cancer Institute, 1-37-1 Kami-lkebukuro, Toshima-ku, Tokyo 170, Japan

Abstract

The APC (adenomatous polyposis coli) gene is responsible for famil- ial adenomatous polyposis and is also associated with the development of sporadic tumors of the colon and stomach. To investigate whether or not mutations of APC play any role in tumors arising in other organs, we examined somatic mutations of this gene in sporadic (nonfamilial) renal cell carcinomas, hepatocellular carcinomas, and cancers of the lung and pancreas. DNAs isolated from tumors were examined by means of a RNase protection analysis, coupled with the polymerase chain reaction followed by DNA sequencing of the polymerase chain reaction products. By screening a part of the APC coding region, we detected somatic mutations in four of ten pancreatic cancers; each of these mutations would yield a truncated .4PC product due to a 1- or 5-base pair deletion. These results imply that mutations in APC contribute to carcinogenesis in the pancreas.

Introduction

The gene responsible for familial adenomatous polyposis, termed A P C 3 (1_4), was isolated in 1991. A P C is considered to be a tumor suppressor gene for the following reasons: (a) LOH at the A P C locus has been observed frequently in colorectal and gastric tumors (5, 6); and (b) somatic mutat ions of A P C have been identified in sporadic forms of these tumors (2, 7, 8). Al though the somatic mutat ions reported thus far were detected only in tumors arising in the gastrointestinal tract, whether A P C plays a role in the proliferation of cells in other organs is still an open question. Frequent L O H at loci on chromosome 5q, where A P C is located, have been reported in renal cell carcinomas (9, 10), hepatocellular carcinomas (11, 12) and can- cers of the lung (13-15); however, that A P C is the gene respon- sible for development of these types of tumor has not been proved. To address these questions, we have examined the mu- tat ion cluster region (MCR) (7) of A P C for somatic mutat ions in sporadic tumors from kidney, liver, lung, and pancreas which represent a common cancer with poor prognosis. The results we report here contribute significantly to a growing understanding of the role of the A P C gene in human disease.

Materials and Methods

DNA Preparation. A total of 84 primary tumor tissues, consisting of 14 renal cell carcinomas, 5 hepatocellular carcinomas, 55 cancers of the lung (1 large cell carcinoma, 5 adenosquamous carcinomas, 36 adenocarcinomas, and 13 tumors of unknown histopathological diag- nosis), and 10 pancreatic cancers (7 well differentiated adenocarcino- mas, 2 moderately differentiated adenocarcinomas, and 1 poorly dif- ferentiated adenocarcinoma) were analyzed in this study. In each case,

Received 8/24/92; accepted 10/13/92. The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work was supported in part by the Ministry of Education, Culture and Science of Japan and in part by the Vehicle Racing Commemorative Foundation.

2 To whom requests for reprints should be addressed. 3 The abbreviations used are: APC, adenomatous polyposis coli; PCR, I)O13,-

merase chain reaction; LOH, loss of heterozygosity.

corresponding noncancerous tissues, either from surrounding normal tissues or from peripheral WBC, were collected. All of the pancreatic DNA samples, from both tumor and normal tissues, were obtained from tissues attached to glass slides in slices 10 ~m thick which had been fixed in formalin and embedded in paraffin; under a microscope, cells were excised from ten slides of each tissue specimen and pooled (16). Other tissues were obtained during surgery and were frozen im- mediately in liquid nitrogen and stored at -80~ until use. Genomic DNAs from tissues attached to glass slides were extracted as described elsewhere (17) and from other tissues as described by Sato et aL (18).

PCR Amplification. DNA sequence corresponding to certain parts of the coding region of APC were amplified by PCR (19). Primer sets for PCR amplification were described previously (20).

RNase Protection Analyses. To detect mutations efficiently, RNase protection analyses were performed according to the method of Winter et al. (21) with some modification (22). In brief, PCR products were hybridized with 32p-labeled RNA transcripts which represent normal APC sequence and digested by an RNase A (Boehringer Mannheim GmbH, Mannheim, Germany) at a final concentration of 25 ~g/ml. This enzyme cleaves RNA at mismatches within DNA-RNA hybrids. Digested products were electrophoresed in an 8% polyacrylamide-8 M urea gel and autoradiographed to detect any extra bands. RNA tran- scripts corresponding to each strand of genomic DNA were examined to increase the efficiency and accuracy of this method.

DNA Sequencing. PCR products which showed different patterns by RNase protection analyses were subsequently sequenced. Template DNAs for the sequencing reactions were prepared either by asymmet- rical PCR (23) or by purification of pooled DNA from at least 50 subclones which contained the PCR product at the EcoRV site of the pBluescript II SK (-) (Stratagene, La Jolla, CA). Sequencing reactions on both strands were performed (24, 25) to confirm mutations.

Results

We first examined expression of A P C in several normal tis- sues by means of reverse t ranscr ipt ion-PCR assays. Expression of actin was also examined as a control of RNA preparation. We detected expression of A P C in a broad spectrum of organs including kidney, liver, lung, and pancreas (data not shown). Then, parts of A P C were amplified by PCR, and the products were analyzed by a RNase protection assay. Results of abnormalit ies detected by the RNase protection analysis are shown in Fig. 1. Samples in which extra bands were detected were then sequenced to determine the specific DNA alterations. Because the amount of DNA was limited for some samples, it was impossible to examine the entire coding region of this very large gene; therefore, we first screened the samples for somatic mutat ions within the "muta t ion cluster region" of A P C (be- tween codons 1286 and 1513). This nearly 700-base pair region is the site where more than two-thirds of the somatic mutat ions in colorectal tumors were observed to date (7). Then, we ex- tended the region for screening in samples where the amount of available DNAs permitted. Table 1 summarizes this informa- tion.

Somatic mutat ions were detected in pancreatic cancers, whereas no mutat ion has thus far been detected in other type of tumors. In four of ten pancreatic cancers where mutat ions were

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APC MUTATIONS IN PANCREATIC CANCER

PN9 PT9 PNIO PTIO . . . . . .

Fig. 1. Results of RNase protection analysis. Abnormal patterns were detected in each lane of tumor, which indicated the presence of somatic mutations. PN, and PT, DNA samples obtained from normal and tumor tissue, respectively.

Table 1 Summary of screening experiments

Somatic Origin of Examined area Tumors mutations

tumor (by codons) examined detected

Kidney 279-1666 14 0 Liver 279-1666 5 0 Lung 582-1666 55 0 Pancreas 742-1666 10 4

detected, we proved that they were somatic events by comparing the mutant DNAs with samples isolated from corresponding normal tissues, by means of RNase protection analyses and/or DNA sequencing. In tumor PT9 (well differentiated adenocar- cinoma), we detected a 5-base pair deletion of either GAATT or AATTG from the GGAATTGG sequence at codons 857-859 (Fig. 2,4); this change generated a new stop codon (TGA) im- mediately downstream, due to a frame shift. In tumor PT4 (well differentiated adenocarcinoma), a deletion of C from GCCCA was detected at codon 1006; and in tumors PT6 (moderately differentiated adenocarcinoma) and PT10 (well differentiated adenocarcinoma), a deletion of A from CAAAC was detected at codon 1444. Again, in each case a new stop codon was created immediately downstream. From DNA sequencing results of tumor PT10, bands corresponding to the normal DNA se- quence were vary faint on the sequencing gel, indicating loss of the normal.4PC allele in this tumor (Fig. 2B). These results are summarized in Table 2.

Discussion

In previous studies, we found somatic mutations of,4PC in

the present study, we searched for somatic mutations of APC in sporadic tumors arising in organs outside the gastrointestinal tract. We chose to examine tumors of kidney, liver, lung, and pancreas for the following reasons: (a) frequent LOH at sites on chromosome 5q has been reported in tumors of kidney (9, 10), liver (11, 12) and lung (13-15); (b) tissues of these four organs express the APC gene; and (c) cancers of these four organs are relatively frequent malignancies with poor prognosis.

Among the tumors tested, we could detect somatic mutations only in pancreatic cancers. Until now, reported genetic alter- ations in pancreatic tumors have been limited to point muta- tions of K-ras (16, 26-31) and p53 (32, 33) and loss of expres- sion of DCC (34). In the course of colorectal tumorigenesis, an accumulation of genetic alterations within genes on chromo- somes 5q (,4PC, MCC), 12p (K-ras), 17p (p53), and 18q (DCC)

(A)

(B)

PN9 PT9

A T C T G G T T A A G

G

T c T G G

PN10 PT10

G A T C .A G A T C /! �9

! i G

i "i~TG

Fig. 2. Autoradiographs of sequencing results from part of APC in pancreatic cancer patients. PN, normal pancreatic DNA; PT, tumor DNA. A, tumor PT 9 showed a 5-base pair deletion of either GAATT or AATTG in GGAATTGG (codons 857-859) resulting in a frame shift followed by a stop codon (TGA). B, tumor PT 10 showed a deletion of A in CAAAC at codons 1444-1445, resulting in a frame shift followed by a stop codon (TAA). The sequence of the antisense strand is shown.

Table 2 Somatic mutations of APC in pancreatic cancers

Nucleotide Tumor Codon change Type of mutation

PT4 (W a) 1006 GCCCA-~GCCA 1-base pair deletion PT6 (M) 1444 CAAAC-~CAAC 1-base pair deletion PT9 (W) 857 GGAATTGG-~GGG 5-base pair deletion PT 10 (W) 1444 CAA.AC-~CAAC 1-base pair deletion

with allelic loss

a W, well differentiated tubular adenocarcinoma; M, moderately differentiated nonfamilial colorectal tumors (2, 7) and gastric cancers (8). In tubular adenocarcinoma.

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APC MUTATIONS IN PANCREATIC CANCER

is cons ide red to be crucia l (35). In the p resen t s tudy, we found

soma t i c m u t a t i o n s o f A P C in four o f ten panc rea t i c cancers .

T a k i n g our resul ts and the ev idence f rom prev ious repor t s in to cons ide ra t ion , it appears tha t c o m b i n a t i o n s o f genet ic alter-

a t ions in A P C , K-ras, p53, and D C C could be i m p o r t a n t for

t umor igenes i s in the pancreas , as they are in the colon. In these two organs , the same or s imi la r mechan i sm(s ) m a y exist in the course o f ca rc inogenes i s .

N o soma t i c A P C m u t a t i o n has thus far been de tec ted in the o the r t u m o r s tes ted here , a l t h o u g h all o f 14 renal cell car- c i n o m a s and 5 hepa toce l lu l a r c a r c i n o m a s and 7 o f 55 lung

cancers had lost one allele at this locus on c h r o m o s o m e 5q (8 -10 , 15). S ince we did no t e x a m i n e the ent i re cod ing region

of A P C , o the r domain ( s ) o f the gene could be closely asso- c ia ted wi th t u m o r s in these types o f tissue. H o w e v e r , we th ink

it is m o r e l ikely tha t a t u m o r suppres so r gene(s) o the r t han A P C , but also loca ted on c h r o m o s o m e 5q, m a y be respons ib le for these t umors . F u r t h e r s tudy will be requ i red to address tha t ques t ion .

A c k n o w l e d g m e n t s

The authors thank Kiyoshi Noguchi (Cancer Institute, Tokyo) for assistance in the preparation of the manuscript.

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1992;52:6696-6698. Cancer Res   Akira Horii, Shuichi Nakatsuru, Yasuo Miyoshi, et al.   Pancreatic Cancer

Gene in HumanAPCFrequent Somatic Mutations of the

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