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[Cancer Research 60, 546-548, February 1, 2000]
© 2000 American Association for Cancer Research

Advances in Brief

No Evidence of Peutz-Jeghers Syndrome Gene LKB1 Involvement in Left-sided Colorectal Carcinomas1

Virpi Launonen, Egle Avizienyte, Anu Loukola, Päivi Laiho, Reijo Salovaara, Heikki Järvinen, Jukka-Pekka Mecklin, Asuka Oku, Miyuki Shimane, Hee C. Kim, Jin C. Kim, Jun-ichi Nezu and Lauri A. Aaltonen2

Departments of Medical Genetics [V. L., E. A., A. L., P. L., L. A. A.] and Pathology [R. S.], Haartman Institute, University of Helsinki, Helsinki FIN-00014, Finland; Second Department of Surgery, Helsinki University Central Hospital, Helsinki, FIN-00290 Finland [H. J.]; Department of Surgery, Central Hospital of Jyväskylä, FIN-40620 Jyväskylä, Finland [J-P. M.]; University of Ulsan College of Medicine and Asan Medical Center, Seoul 138-736, Korea [H. C. K., J. C. K.]; and Gene Search Program, Chugai Research Institute For Molecular Medicine, Inc., Ibaraki 300-4101, Japan [A. O., M. S., J-i. N.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 
LKB1 serine/threonine kinase is a gene for Peutz-Jeghers cancer predisposition syndrome. Most studies have detected a low frequency of LKB1 defects in sporadic cancer. A notable exception is a recent report describing frequent, mostly missense type, LKB1 mutations in Korean distal colorectal tumors. To clarify the role of LKB1 in colon cancer, we scrutinized 50 left-sided Korean and Finnish specimens. No somatic mutations were found. The seven Korean somatic missense mutations reported previously were functionally analyzed, and five were found not to alter LKB1 kinase activity. One of these changes was found to be a germ-line polymorphism. LKB1 involvement in distal colorectal cancer is not common.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results and Discussion
 REFERENCES
 
Germ-line mutations of the serine/threonine kinase gene LKB1 cause PJS3 (1 , 2) , which is characterized by hamartomatous polyposis of the gastrointestinal tract and mucocutaneous pigmentation (3, 4, 5) . Patients with PJS are also known to have a significantly increased risk of cancer as compared with the normal population (6 , 7) . A wide spectrum of neoplasia, particularly cancers of the gastrointestinal tract (stomach, small intestine, and colon), pancreas, breast, testis, and ovary appears to be associated with PJS (5, 6, 7) .

Most of the detected germ-line LKB1 mutations lead to truncated protein products (1 , 2 , 8 , 9) . Loss-of-function type mutations and the observation of loss of heterozygosity in polyps of PJS patients indicate that LKB1 acts as a tumor suppressor gene (10) , and further studies have confirmed this notion (9 , 11 , 12) . These findings indicate that LKB1 might possibly be involved in the pathogenesis of sporadic cancers as well. However, somatic mutations appear to be rare, at least in breast, colorectal, gastric, testicular, pancreatic, and ovarian cancer, as well as in malignant melanoma (13, 14, 15, 16, 17, 18, 19, 20) . A notable exception has been a recent report by Dong et al. (21) . These authors had detected a high frequency of somatic mutations in Korean left-sided colorectal tumors (10 of 19, 53.8%) and left-sided adenomas with high-grade dysplasia (2 of 7, 28.6%), highlighting LKB1 as an important gene contributing to sporadic colorectal tumorigenesis.

Because we and others (14 , 16 , 17) had found little evidence for LKB1 involvement in sporadic colorectal cancer, we investigated a series of 21 Finnish and 29 Korean distal colorectal cancer samples for LKB1 mutations to clarify the role of this gene in colonic tumor initiation and progression. Furthermore, we analyzed the functional consequences of the seven missense mutations that were reported previously in left-sided colon cancer (21) to evaluate their possible effect on LKB1 kinase function.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results and Discussion
 REFERENCES
 
A series of 21 Finnish and 29 Korean left-sided sporadic colorectal carcinomas were obtained for LKB1 screening. Previous work had proposed that LKB1 mutations are not an early change in colorectal tumorigenesis (21) . Thus, in this work, it was ensured that a considerable proportion of the lesions were well advanced, although no other selection criteria were introduced. The stage distribution of the samples according to Dukes’ classification was: A, 3 (6%); B, 10 (20%); C, 20 (40%); and D, 17 (34%). Mutation analyses were typically performed using direct genomic sequencing; the original goal was to analyze approximately 20 Finnish and 20 Korean cases using this resource-demanding method (final numbers, 21 and 23, respectively). SSCP was used to evaluate the remaining six Korean samples. The conditions for SSCP and sequencing are described by Avizienyte et al. (14 , 15) .

To further evaluate an LKB1 missense variant (F354L) present in both of the studied populations, ASO was performed. Genomic DNA samples were amplified either with primers used in SSCP or direct sequencing. PCR products were run in 2% agarose (NuSieve) gel to verify the amplification, and thus avoiding the need for hybridization with a wild-type probe. PCR products from three individuals were pooled together. Slot-blots were made on nylon membranes using a vacuum manifold. Probe was labeled with [{gamma}-32P]dATP using T4-polynucleotide kinase (New England BioLabs). Filters were hybridized at 62°C with a probe containing the mutant sequence (5'-GGA CCT CTT GGA CAT CGA G-3'). If a positive signal was obtained, the respective samples were rehybridized separately on a new membrane. Positive results were verified by genomic sequencing.

Altogether 299 normal tissue DNA specimens from Finnish colorectal carcinoma patients and 84 anonymous cancer-free blood donors were analyzed. To evaluate the frequency of the alteration in the Korean population, normal tissue DNA derived from 50 colorectal cancer patients and 36 cancer-free blood donors were obtained for ASO screening.

The protein kinase activity studies of four missense type mutations were based on an autophosphorylation assay that has been described previously (22) . The expression plasmid DNA, pcDNA3/LKB1 myc, which contained the wild-type LKB1 coding sequence and c-Myc epitope tag (EQKLISEEDL) on its COOH terminus, was constructed as described previously (11) . Using this plasmid DNA as template, each of the mutant LKB1 expression plasmids was generated by in vitro mutagenesis using the GeneEditor in vitro site-directed mutagenesis system (Promega), according to the procedure suggested by the manufacturer. The data on mutagenic oligonucleotides used is available on request.


   Results and Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
REFERENCES
 
A total of 21 Finnish left-sided colorectal carcinomas were first screened for LKB1. In this series, only one missense change was detected in the coding sequences and exon/intron boundaries. All of the other changes were previously reported intronic polymorphisms. The observed missense change converts phenylalanine to leucine at codon 354 (F354L). This variant was also seen in normal tissue of the patient, indicating a germ-line alteration (Fig. 1)Citation . Thus, no somatic alterations were detected. Interestingly, the germ-line missense change was identical to one of the somatic mutations reported by Dong et al. (21) .



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  		Fig. 1. Heterozygous LKB1 missense germ-line variant detected in Finnish and Korean populations [F354L (C->G); arrow].

 
To further evaluate the missense variant, we performed normal tissue DNA ASO screening of 299 unselected Finnish colorectal carcinoma patients. None of these displayed the change. Taken together, only 1 of 320 (0.3%) Finnish colorectal carcinoma patients displayed F354L. For this variant, we also analyzed 84 Finnish cancer-free controls, and none of these carried the change.

Somatic mutations in LKB1 coding region were not detected in the studied 29 Korean distal colorectal carcinoma cases. In addition to intronic polymorphisms, four samples displaying heterozygous F354L missense type change (14%), presumably a germ-line variant as in one of the Finnish patients, were detected. To confirm the presence of this germ-line variant in the Korean population, we screened normal tissue DNA from 50 additional colorectal cancer patients and 36 cancer-free individuals for germ-line LKB1 F354L by ASO. Heterozygous F354L (confirmed by sequencing) was observed in one carcinoma patient and in two controls (Fig. 2)Citation . The overall frequency of F354L variant in Korean colorectal carcinoma patients was 6.3% (5 of 79) and 5.6% (2 of 36) in Korean cancer-free controls. This result suggests that the variant is a neutral polymorphism.



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  		Fig. 2. F354 (C->G) detected using ASO. A positive control (1) was included for all hybridizations. In the first round, three samples were pooled together, and samples from positive pools (4) were subsequently hybridized individually. Finally, samples positive by ASO were sequenced to confirm the presence of heterozygous F354 (C->G).

 
We next studied protein kinase activity of the seven missense changes (G171S, E199K, D208N, G215D, P281L, F354L, and T367 M) reported as somatic mutations in a previous study focusing on Korean left-sided colorectal carcinomas (Ref. 21 ; Fig. 3Citation ). Unexpectedly, in the autophosphorylation assay, normal activity was observed in five of seven of the missense type mutations, suggesting that these changes do not alter the kinase activity of LKB1. Five of the changes, G171S, E199K, D208N, G215D, and P281L, are located inside of the catalytic kinase domain region, and amino acids 171, 199, 208, 215, and 367 are conserved among human, mouse, and Xenopus.



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  		Fig. 3. The protein kinase activity assay of the seven previously reported missense changes. Five variants (G171S, E199K, D208N, F354L, and T367M) displayed normal kinase function. K78I served as a negative control (kinase-dead mutant).

 
The majority of the reported somatic LKB1 mutations are missense type of changes (13, 14, 15, 16, 17, 18, 19, 20) . Before this work, F354L change had been reported only once, by Dong et al. (21) . In their study, the frequency of this particular change was 2% (1 of 49). On the basis of the results of this study, the missense change F354L appears to be a polymorphism, which occurs relatively frequently (7 of 115, 6%) in the Korean population. That only 1 of 404 studied Finns (0.2%) carried the change is well compatible with the fact that this change has not been detected in previous LKB1 studies focusing on cancer in Caucasian populations. Further studies should clarify whether this change is associated with cancer predisposition, but the present data suggest that this is unlikely.

Only two of seven missense type variants abolished the kinase activity of LKB1, demonstrated by the autophosphorylation assay. Little is known about LKB1 function, and changes with no effect on autophosphorylation may have significance in view of putative other functions. However, the complete absence of somatic mutations in the studied series of distal colorectal cancers, normal kinase function of five of the previously reported seven aberrant alleles, and presence of one of the aberrant alleles as a germ-line variant in normal populations all argue against a prominent role of LKB1 in colorectal cancer. It is likely that some somatic LKB1 mutations occur in colorectal tumors, but as in all other tumor types studied thus far, the frequency of these defects appears to be low.


   ACKNOWLEDGMENTS
 
We thank Annika Lahti, Kirsi Laukkanen, Siv Lindroos, and Sinikka Lindh for technical assistance.


   FOOTNOTES
 
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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant BMH-CT98-3865 from the European Commission and grants from the Academy of Finland, Finnish Cancer Society, Sigrid Juselius Foundation, Helsinki University Central Hospital, and Biocentrum Helsinki. Back

2 To whom requests for reprints should be addressed, at Department of Medical Genetics, Haartman Institute, P. O. Box 21, University of Helsinki, Helsinki FIN-00014, Finland. Back

3 The abbreviations used are: PJS, Peutz-Jeghers syndrome; ASO, allele-specific oligonucleotide hybridization; SSCP, single strand conformation polymorphism. Back

Received 10/15/99. Accepted 12/14/99.


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 Materials and Methods
 Results and Discussion
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