Oncogene (2005) 24, 4588–4590
& 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc
SHORT REPORTS
Genetic alterations of the KLF6 gene in gastric cancer Yong Gu Cho1, Chang Jae Kim1, Cho Hyun Park2, Young Mok Yang3, Su Young Kim1, Suk Woo Nam1, Sug Hyung Lee1, Nam Jin Yoo1, Jung Young Lee1 and Won Sang Park*,1 1
Department of Pathology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea; 2Department of Pathology Surgery, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea; 3Genetic Laboratory of Premedical Course, Kon-Kuk University College of Medicine, Chungju 380-701, Korea
The KLF6 is a zinc-finger tumor suppressor that is frequently mutated in several human cancers and broadly involved in differentiation and development, growthrelated signal transduction, cell proliferation, apoptosis, and angiogenesis. To determine whether genetic alterations of KLF6 gene are involved in the development and/or progression of gastric cancer, we have screened a set of 80 sporadic gastric cancers for mutations and allele loss of the KLF6 gene. Four missense mutations, S155R, P172 T, S180L, and R198 K, were detected in transactivation domain of the KLF6 gene and one of them had biallelic mutations with somatic mutation of one allele and loss of the remaining allele. All of the cases with mutation were of advanced intestinal-type gastric cancer with lymph node metastasis. In addition, 16 (43.2%) of 37 informative cases showed allelic loss at KLF6 locus. Interestingly, allelic loss was also frequent in intestinal-type gastric cancer. Therefore, our data suggest that genetic alterations of KLF6 gene might play an important role in the development or progression of sporadic gastric cancers. Oncogene (2005) 24, 4588–4590. doi:10.1038/sj.onc.1208670 Published online 11 April 2005 Keywords: mutation; tumor suppressor gene; loss of heterozygosity
Gastric cancer is the most frequent malignancy of the gastrointestinal tract in Korean and certain Southeast Asian populations and one of the leading causes of cancer death in the world, accounting for an estimated 10% of all malignancies worldwide (Parkin et al., 1993). In Korea, it accounts for an estimated 20.9% of all malignancies, with 24.4% in the male population and 16.3% in the female population (Suh et al., 2000). However, little is known about the molecular genetic event in its development and progression. Kruppel-like factor 6 (KLF6) is a ubiquitously expressed zinc-finger transcription factor that is part of a growing KLF family. The KLF family is broadly involved in differentiation and development, growthrelated signal transduction, cell proliferation, apoptosis, *Correspondence: WS Park; E-mail:
[email protected] Received 20 December 2004; revised 23 February 2005; accepted 24 February 2005; published online 11 April 2005
and angiogenesis (Bieker, 2001; Black et al., 2001). Recently, KLF6 gene mutations have been identified in hepatocellular carcinoma, prostate, and colon cancers (Narla et al., 2001; Chen et al., 2003; Kremer-Tal et al., 2004; Reeves et al., 2004). Wild-type KLF6 upregulates the cell cycle inhibitor p21 in a TP53-independent manner and suppresses growth, whereas tumor-derived KLF6 mutants fail to upregulate p21 or suppress proliferation in prostatic and non-small cell lung cancer cell (Narla et al., 2001; Ito et al., 2004). These findings suggest the possibility of a tumor suppressor role of KLF6 in cancer pathogenesis. Allelotype studies have shown that specific regions are frequently deleted in many tumor types and frequent loss of heterozygosity (LOH) at a certain chromosomal region is a hallmark of the existence of a tumor suppressor gene. Therefore, the identification of consistent areas of chromosomal deletion in particular tumor DNA may indicate regions harboring tumor suppressor gene at this locus, which contributes to carcinogenesis at that tissue. In previous allelotype analysis on whole chromosomes of gastric cancer, frequent LOH has been reported on the short arm of chromosome 10 (Tamura et al., 1996; Nishizuka et al., 1998; Yustein et al., 1999; Hiyama et al., 2003). Because the KLF6 gene is located on chromosome 10p15 (Onyango et al., 1998) and efforts to assess the impact of somatic KLF6 inactivation on cancer behavior could give us important pathogenetic and prognostic information, we screened 80 sporadic gastric cancers for mutations and allelic loss of the KLF6 gene. A total of 80 methacarn-fixed gastric cancer specimens were obtained from the College of Medicine, The Catholic University of Korea in Seoul, Korea. A total of 42 cases were of intestinal-type and 38 were of diffusetype gastric cancers. Informed consent was obtained from every patient. No patient had a family history. We searched for potential mutations in all four exons for the KLF6 gene in 80 gastric cancer tissues by PCR–SSCP and sequencing analysis. When a band shift was observed in a sample, the SSCP procedure including PCR was repeated to verify the band shift and the PCR products were subject to DNA sequencing. We detected four mutations in exon 2 of KLF6 gene. All of the mutations were verified through triplicate experiments including tissue microdissection, PCR, SSCP, and direct sequencing analysis to ensure the specificity of the
Mutations of the KLF6 gene in gastric cancer YG Cho et al
4589
results. As shown Figure 1, all of the mutations were missense mutations: a C to A transversion at nucleoides 3412 and 3461 (Ser to Arg at codon 155 and Phe to The at codon 172, respectively), a C to T transition at nucleotide 3486 (Ser to Leu at codon 180), and a G to A transition at nucleotide 3541 (Arg to Lys at codon 198). Interestingly, two of the mutations affected serine residues and one mutation affected the amino-acid
Figure 1 Representative results showing SSCP and direct sequencing analyses of the KLF6 gene in gastric cancer. We selectively procured tumor cells from Hematoxyln- and eosin-stained slides using a laser microdissection device (ION LMD, JungWoo International Co, Seoul, Korea) and inflammatory or surrounding normal mucosal cells for corresponding normal DNAs from the same slides in all cases. DNA extraction was performed by a modified single-step DNA extraction method, as described previously (Lee et al., 1998). Genomic DNAs from each cancer cell and corresponding noncancerous gastric mucosa tissue were amplified with seven sets of primers covering the entire coding region (exons 1–4) of the KLF6 gene , which were described previously (Ito et al., 2004). Numbering of DNA of the KLF6 gene was carried out with respect to the ATG start codon according to the genomic sequence of GenBank accession no. NT_077567. Each PCR reaction was performed under standard conditions, as previously described (Lee et al., 1998). SSCP of DNAs from tumors of case no. 24 showed only two aberrant bands without any wild-type bands as compared to SSCP from corresponding normal tissue, and the rest demonstrated one aberrant band with wild-type bands. The mutations, (a) AGC to AGA (S155R), (b) CCA ACA (P172 T), (c) TCG to TTG (S180L), and (d) AGG - AAG (R198 K) in exon 2 of the gene, are represented. (N, normal; T, Tumor)
residues next to a serine. The mutations are present in tumor tissue, but absent in corresponding normal tissue, suggesting somatic mutation. In addition, one of the cases with mutation (case no. 24) showed only aberrant migrating mutant bands with absence of wild-type bands, indicating mutation of one allele and loss of the remaining allele (Figure 1). Next, to identify the expression level of KLF6 protein in the four cases with mutation, we performed immunohistochemical analysis on paraffin-embedded tissues with the anti-KLF6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Whereas KLF6 expression was observed mainly in the cytoplasm and nucleus of normal glandular epithelium and inflammatory cells, reduced expression of KLF6 protein in cancer cells was seen in three of four cases with mutation (data not shown). Clinically, all of the cases with mutation are of advanced intestinal-type gastric cancer with surrounding lymph node metastasis, and two of them died of cancer. We also analysed allelic loss of KLF6 with KLF6M1 and KLF6M2 markers, which is located no more than 40 kb from the KLF6 locus. Of 80 gastric cancers, microsatellite instability (MSI) was seen in nine and two cases at KLF6M1 and KLF6M2 markers, respectively (data not shown). A total of 25 (35.2%) of 71 cases without MSI at KLF6M1 marker were informative and LOH was found in 10 (40.0%). For KLF6M2 marker, 21 of 78 cases without MSI were informative and six (28.6%) of them showed allelic loss. A total of 37 cases were informative at KLF6M1 and/or KLF6M2 markers, and 16 (43.2%) of them showed allelic loss (data not shown). Interestingly, one of the cases (case no. 24) with KLF6 mutation showed LOH, suggesting biallelic inactivation through missense mutation of one allele and loss of the remaining allele. Two of the cases with mutation showed retention of the retained allele and one case was noninformative at the above markers, but the presence of wild-type band on SSCP gel led us to interpret the allelic status as retention of both mutantand wild-type alleles. Histologically, 13 of 16 cases with allelic loss were of intestinal-type gastric cancer and three cases were diffuse type, and allelic loss at KLF6 locus is more frequent in intestinal-type gastric cancer (w2 test, P ¼ 0.01). Finally, our data suggest that genetic alterations of the KLF6 gene might play an important role in the development or progression of gastric cancer, especially in intestinal-type gastric cancer. Given previous finding suggesting inactivation of KLF6 in hepatocellular carcinoma by loss of one KLF6 allele (Kremer-Tal et al., 2004), it is possible that haploinsufficiency of the KLF6 gene, like other tumor suppressor genes (Fodde and Smits, 2002), may lead to gastric cancer development or progression. However, we cannot completely rule out the possibility of a genetic alteration in other regions, such as the promoter, noncoding exons, and splice sites, and promoter methylation (Yamashita et al., 2002). In addition, our results may underestimate the prevalence of the KLF6 somatic mutations in gastric cancer, as the sensitivity rate of SSCP analysis for the detection of single-base substitutions is estimated at 80% (Sheffield et al., 1993). Oncogene
Mutations of the KLF6 gene in gastric cancer YG Cho et al
4590
Further efforts are needed to elucidate the role of other mechanisms of KLF6 inactivation in gastric cancers. The KLF family is intimately linked to growth-related changes in transcription as evidenced by changes in activity and expression of family members with cell growth and by their participation in a variety of mitogenic and growth-inhibitory signal transduction pathways. The expression and genetic alterations in the family members seen with tumor progression, together with their interaction with multiple oncogenes and tumor suppressors, indicate that KLF family members play an important role in the development of tumors (Black et al., 2001; Narla et al., 2001). Recently, KLF6 gene mutations have been identified in hepatocellular carcinoma, prostate, and colon cancers, and most mutations have been found in exon 2 encoding three-fourths of wild-type protein (Chen et al., 2003; Kremer-Tal et al., 2004; Reeves et al., 2004). In the present study, we found four somatic missense mutations, S155R, P172 T, S180 K, and R198 K, which were confined to the activation domain of the KLF6 gene. Interestingly, two mutations at serine residue and one mutation at the amino-acid residues next to a serine further support that serine residues in the transactivation domain of KLF6 may be important for its function
(Jeng and Hsu, 2003). In particular, mutation of serine residue at codon 155 is very common in several human cancers, and is predicted to affect consensus phosphorylation sites for GSK3b, a Wnt signaling regulator whose role in gastric cancer is well established (Clements et al., 2002). Functional studies show that wild-type KLF6 upregulates p21 (WAF1/CIP1) in a p53-independent manner and suppresses cell proliferation and tumor growth via induction of apoptosis (Narla et al., 2001; Ito et al., 2004). Furthermore, KLF6 inhibits cyclin D1/ cdk4 activity and promotes G1 cell cycle arrest (Benzeno et al., 2004). Although we did not examine the functional analysis of these mutant proteins, it is likely that KLF6 mutations detected in this study may also cause the defect in growth suppressive activity of KLF6 and may contribute to the development or progression of gastric cancer. Additional studies of large patient populations are needed to verify these initial observations and will broaden our understanding of the role KLF6 and gastric carcinogenesis. Acknowledgements This work was supported by the Korea Science & Engineering Foundation (KOSEF) through the Cell Death Disease Research Center at The Catholic University of Korea.
References Benzeno S, Narla G, Allina J, Cheng GZ, Reeves HL, Banck MS, Odin JA, Diehl JA, Germain D and Friedman SL. (2004). Cancer Res., 64, 3885–3891. Bieker JJ. (2001). J. Biol. Chem., 276, 34355–34358. Black AR, Black JD and Azizkhan-Clifford J. (2001). J. Cell. Physiol., 188, 143–160. Chen C, Hyytinen E-R, Sun X, Helin HJ, Koivisto PA, Frierson Jr HF, Vessella RL and Dong J-T. (2003). Am. J. Pathol., 162, 1349–1354. Clements WM, Wang J, Sarnaik A, Kim OJ, MacDonald J, Fenoglio-Preiser C, Groden J and Lowy AM. (2002). Cancer Res., 62, 3503–3506. Fodde R and Smits R. (2002). Science, 298, 761. Hiyama T, Tanaka S, Shima H, Kose K, Kitadai Y, Ito M, Sumii M, Yoshihara M, Shimamoto F, Haruma K and Chayama K. (2003). Oncol Rep., 10, 1297–1299. Ito G, Uchiyama M, Kondo M, Mori S, Usami N, Maeda O, Kawabe T, Hasegawa Y, Shimokata K and Sekido Y. (2004). Cancer Res., 64, 3838–3843. Jeng Y-M and Hsu H-C. (2003). Int. J. Cancer, 105, 625–629. Kremer-Tal S, Reeves HI, Narla G, Thung SN, Schwartz M, Difel A, Katz A, Bruix J, Bioulac-Sage P, Martignetti JA and Friedman SL. (2004). Hepatology, 40, 1047–1052. Lee JY, Dong SM, Kim SY, Yoo NJ, Lee SH and Park WS. (1998). Virchows Arch., 433, 305–309.
Oncogene
Narla G, Heath KE, Reeves HL, Li D, Giono LE, Kimmelman AC, Glucksman MJ, Narla J, Eng FJ, Chan AM, Ferrari AC, Martignetti JA and Friedman SL. (2001). Science, 294, 2563–2566. Nishizuka S, Tamura G, Terashima M and Satodate R. (1998). J. Pathol., 185, 38–43. Onyango P, Koritschoner NP, Patriot LC, Zenke M and Weith A. (1998). Genomics, 48, 143–144. Parkin DM, Pisanin P and Ferlay J. (1993). Int. J. Cancer, 54, 594–606. Reeves HL, Narla G, OgunbiYi O, Haq AI, Katz A, Benzeno S, Hod E, Harpaz N, Goldberg S, Tal-Kremer S, Eng FJ, Arthur MJ, Martignetti JA and Friedman SL. (2004). Gastroenterology, 126, 1090–1103. Sheffield VC, Beck JS, Kwitek AE, Sandstrom DW and Stone EM. (1993). Genomics, 16, 325–332. Suh CI, Suh K-A, Park S-H, Chang HJ, Ko J-W and Ahn D-H. (2000). J. Korean Cancer Assoc., 32, 827–834. Tamura G, Sakata K, Nishizuka S, Maesawa C, Suzuki Y, Terashima M, Eda Y and Satodate R. (1996). J. Pathol., 180, 371–377. Yamashita K, Upadhyay S, Osada M, Hoque MO, Xiao Y, Mori M, Sato F, Meltzer SJ and Sidransky D. (2002). Cancer Cell, 2, 485–495. Yustein AS, Harper JC, Petroni GR, Cummings OW, Moskaluk CA and Powell SM. (1999). Cancer Res., 59, 1437–1441.
