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Chk1 frameshift mutation in sporadic and hereditary non-polyposis colorectal cancers with microsatellite instability
EJSO 33 (2007) 580e585
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Chk1 frameshift mutation in sporadic and hereditary non-polyposis colorectal cancers with microsatellite instability C.J. Kim a, J.H. Lee a, J.W. Song, Y.G. Cho, S.Y. Kim, S.W. Nam, N.J. Yoo, W.S. Park, J.Y. Lee* Department of Pathology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Republic of Korea Accepted 15 February 2007 Available online 3 April 2007
Abstract Aim: Protein kinase Chk1 (hChk1) is essential in human cells for cell cycle arrest in response to DNA damage, and has been shown to play an important role in the G2/M checkpoint. The BRAF mutations have been suggested to be linked with defective mismatch repair in colorectal cancers. The aim of this study was to investigate whether a frameshift mutation within the Chk1 gene contribute to the development or progression of eastern sporadic and hereditary non-polyposis colorectal cancer (HNPCC) with microsatellite instability (MSI). Methods: We analyzed MSI using the 6 microsatellite markers and a frameshift mutation in the BRAF gene and in poly(A)9 within the Chk1 gene in 51 sporadic colorectal cancer and 14 HNPCC specimens. Results: Eleven of the 51 sporadic colorectal cancers and all of the 14 HNPCCs were MSI-positive. Chk1 frameshift mutations were observed in 2 and 3 sporadic colon cancers and HNPCC, respectively, whereas no BRAF mutations were detected in these samples. Interestingly, all cases with the Chk1 frameshift mutation had high-frequency MSI. Conclusion: These results suggest that the Chk1 gene is a target of genomic instability in MSI-positive colorectal cancers and that the Chk1 framshift mutations might be involved in colorectal tumourigenesis through a defect in response to DNA damage in a subset of sporadic colorectal cancers and HNPCCs. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Protein kinase Chk1 (CHK1); Microsatellite instability (MSI); BRAF; Mutation; Colorectal cancers
Introduction Microsatellite repeats are widely distributed throughout the genome. The genes for mismatch repair are responsible for eliminating insertion or deletion mutations in these repeat sequences that arise during DNA replication. Inactivation of the genes responsible for mismatch repair induces a deficiency in mismatch repair, which leads to instability of the microsatellite sequence such as poly(A) or poly(CA) repeats and results in a mutator phenotype. Defective mismatch repair is believed to promote tumorigenesis by accelerating the accumulation of genetic alterations in oncogenes and/or tumor suppressor genes. Microsatellite instability (MSI) is a form of genetic instability in virtually * Corresponding author. Tel.: þ82 2 590 1190; fax: þ82 2 537 6586. E-mail address: [email protected] (J.Y. Lee). a Both these authors contributed equally to this work. 0748-7983/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ejso.2007.02.007
all tumors from patients with HNPCC and a subset of various sporadic tumors, including colorectal and gastric cancer.1e5 It was reported that more than 90% of colorectal cancers in HNPCC patients are MSI, whereas only 15% of sporadic colorectal cancers are MSI.6 However, molecular mechanism of MSI in colorectal carcinogenesis is still unclear. The cell has developed screening systems for maintaining the stability of genetic material. These systems detect DNA damage and facilitate its repair by imposing checkpoint arrests on the cell cycle.7 A major checkpoint of the cell cycle occurs at the end of the G1 phase and prior to entry into the S phase. A second major checkpoint exists at the exit from the G2 phase and before the entry into the M phase.8e10 Indeed, one of the hallmarks of cancer cells is a loss of a checkpoint controlling mechanism.11e14 The Chk1 gene, which is a homolog of the Schizosaccharomyces pombe Chk1 protein kinase,15,16 is an important component of the G2 checkpoint.
C.J. Kim et al. / EJSO 33 (2007) 580e585
This gene phosphorylates Cdc25 and prevents the dephosphorylation of Cdc2.9,10,17e19 In this state, the cell cannot undergo mitosis. Interestingly, it has been shown that Chk1 gene sequence contains a tract of nine adenines in its coding region. Moreover, frameshift mutations in this tract have been found in western colorectal and endometrial cancers with a high degree of MSI.20 However, CHK1 frameshift mutations have not been studied in eastern population. Recently, the activating mutations of the BRAF gene have been reported in various cancers, including colorectal cancers21,22 and alterations of BRAF have been implicated in the carcinogenesis of colorectal tumors with MSI.23 However, extremely low frequency of BRAF mutation in eastern HNPCCs has been reported.24 Thus, it is important to investigate whether the BRAF mutations is linked with mismatch repair deficient colorectal cancers in eastern country. This study searched for MSI and mutations of the Chk1 and BRAF genes in 51 Korean sporadic colorectal cancer and 17 HNPCC specimens to determine if a defective mismatch repair systems results in Chk1 and BRAF mutations in eastern patients with colorectal cancer. Materials and methods Tumour specimens A total of 51 formalin-fixed, paraffin-embedded sporadic colon cancers and 14 HNPCC specimens were obtained from the College of Medicine, The Catholic University of Korea. HNPCC cases were located in the right-side colon and sporadic cancers were located in the left-side colon. Tumor masses ranged from 13 to 70 mm on longest diameter, with a mean diameter of 42 mm. Tumor stage was classified according to the Dukes’ criteria.25 There were 8, 24, 30, 3 cases with stage A, B, C, and D, respectively. Histologically, the cancers consisted of 2 well-differentiated, 62 moderately-differentiated, and 1 poorly-differentiated adenocarcinoma. Informed consent was obtained according to the Declaration of Helsinki. The institutional review board of the Catholic University of Korea, College of Medicine, approved this study. Fourteen HNPCC patients fulfilled for the clinical criteria for HNPCC.26 None of the patients with sporadic colorectal cancer had a family history of the disease. The Hematoxylin & Eosin (H & E) stained histological sections from each case were reviewed. Two pathologists screened the histological sections and selected the areas of representative tumor cells. Malignant cells were selectively procured from the H & E stained slides without contamination from normal cells using a laser microdissection device (ION LMD, JungWoo international Co, Seoul, Korea). The normal cells were also obtained from the corresponding cancer-free colonic mucosa. DNA extraction was carried out using a slight modification of single-step DNA extraction method previously described.27
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Immunohistochemistry for the MLH-1 and MSH-2 in HNPCC specimens MLH1 and MSH2 protein expression in HNPCC specimens was measured by immunohistochemistry. Two mm cancer tissue sections were cut the day before use and stained according to standard protocols. To maximize the signal on immunohistochemistry, two strategies were used in the present study, i.e. antigen retrieval in citrate buffer, and signal amplification with biotinylated tyramide. For the former, heat-induced epitope retrieval was conducted by immersing the slides in Coplin jars filled with 10 mmol/L citrate buffer (pH 6.0) and boiling the buffer for 30 min in a pressure cooker (Nordic Ware, Minneapolis, MN, USA) inside a microwave oven at 700 W; the jars were then cooled for 20 min. For the latter, the Renaissance TSA indirect kit (NEN Life Science, Boston, MA, USA), which included streptavidin-peroxidase and biotinylated tyramide, was used. After rinsing with PBS, the slides were treated with 1% H2O2 in PBS for 15 min at room temperature to abolish endogenous peroxidase activity. After washing with TNT buffer (0.1 mol/L TriseHCl, pH 7.4, 0.15 mol/L NaCl and 0.05% Tween 20) for 20 min, the slides were treated with TNB buffer (0.1 mol/L TriseHCl, pH 7.4, 0.15 mol/L NaCl and 0.5% blocking reagent). Sections were incubated overnight at 4 C with the antibody for MLH1 protein (EMD Biosciences, San Diego, CA, USA; 1:100) and MSH2 (EMD Biosciences, San Diego, CA, USA; 1:100). Detection was carried out using biotinylated goat anti-mouse antibody (Sigma, St. Louis, MO, USA), followed by incubation with peroxidase-linked avidin-biotin complex. Diaminobenzidine was used as chromogen, and the slide was counterstained with Mayer’s hematoxylin. The specificity of antibodies was confirmed in 9 cancer cell lines by Western blot analysis (data not shown). The tumors were interpreted as negative when immunostaining was weak, like the corresponding normal colonic mucosa, or when immunopositive cells were less than 30% of the cancer cells. The results were reviewed independently by 2 pathologists. As negative controls, the slide was treated by replacement of primary antibody with non-immune serum. MSI analysis Somatic microsatellite alterations were analyzed by polymerase chain reaction (PCR), as previously reported.28 In order to identify the MSI in cancer, the microsatellite markers, including BAT-25, BAT-26, BAT-34, D2S123, D17S250, and APCII, were recommended by a National Cancer Institute workshop.2 Only those cases showing an unequivocally distinct additional band or shifts in the tumor tissue DNA compared with normal tissue DNA were recorded and classified as MSI. According to the Bethesda criteria, tumors with MSI-positive cases in more than two informative markers were considered to be MSI-H.2
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Chk1 frameshift mutation and BRAF mutation analysis The frameshift mutation in the mononucleotides runs of the Chk1 gene were determined by amplifying genomic DNAs from each cancer cells and corresponding non-cancerous colonic mucosal tissues with the primer set covering the 93-base pair coding region encompassing the Chk1 poly(A)9 tracts. The primer sequence is as follows; 50 CTCGCTGGAGAATTGCCA-30 (sense) and 50 -TTTCCAA GGGTTGAGGTA-30 (antisense). Mutations in exon 11 and 15 of the BRAF gene, which contain mutational hot spots, were also examined. The primer sequences for exon 11 and 15 are as follows; for exon 11, 50 -GGACTCGAGT GATGATTGG-30 (sense) and 50 -GTGGTGACATTGTGA CAAGTC-30 (antisense) and for exon 15, 50 -TTCCT TTACTTACTACACCTCA-30 (sense) and 50 -GTTGTCTG GATCCATTTTGT-30 . Each polymerase chain reaction (PCR) procedure was performed under standard conditions in a 10 ml reaction mixture containing 1 ml of template DNA, 0.5 mM of each primer, 0.2 mM of each deoxynucleotide triphosphate, 1.5 mM MgCl2, 0.4 unit of Ampli Taq
gold polymerase (Perkin-Elmer, Foster City, CA), 0.5 mCi of [32P]dCTP (Amersham, Buckinghamshire, UK), and 1 ml of 10 buffer. The reaction mixture was denatured for 12 min at 94 C and incubated for 35 cycles (denaturing for 40 s at 94 C, annealing for 40 s at 50e56 C and extension for 40 s at 72 C). A final extension step was carried out for 5 min at 72 C. After amplification, the PCR products were denatured for 5 min at 95 C in a 1:1 dilution of a sample buffer containing 98% formamide/5 mmol/L NaOH. The products were loaded onto a single SSCP gel (FMC Mutation Detection Enhancement system; Intermountain Scientific, Kaysville, UT) with 10% glycerol. After electrophoresis, the gels were transferred to 3 MM Whatman paper and dried. Autoradiography was then performed using Kodak X-OMAT film (Eastman Kodak, Rochester, NY). When a band shift was observed in a sample, it was verified by repeating the SSCP procedure including PCR and subjecting the PCR products to DNA sequencing. All mutations were verified through triplicate experiments including tissue microdissection, PCR, SSCP, and direct sequencing analysis to ensure the specificity of the results.
Figure 1. Autoradiograms of MSI analysis in HNPCC case No. 13 are shown for each of the 6 MSI markers. The case shows MSI at all 6 loci (BAT-26, BAT-25, BAT-34, D2S123, D17S250, and APCII ). The arrowheads indicate MSI in the tumor DNA. N, normal DNA; T, tumor DNA.
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Results MSI in colorectal cancers MSI-positive cases were found in 11 out of 51 sporadic colorectal cancers and all of the HNPCCs, respectively. Interestingly, 12 of the HNPCC cases showed widespread high frequency MSI (MSI-H), whereas only 4 sporadic colon cancers showed MSI-H. Considering the pattern of MSI, all high frequency MSI cases tended to be MSI-positive in the BAT series. Fig. 1 gives representative examples of MSI. Immunohistochemistry in HNPCCs Normal colonic mucosa showed moderate to strong expression of MLH1 and MSH2 proteins mainly in the nucleus of the glandular epithelial cells. Expression of MLH1 and MSH2 in tumor cells was compared with their corresponding normal colonic mucosa. Finally, loss of expression of MLH1 and MSH2 proteins was detected in 7 (50%) of 14 HNPCCs (Fig. 2). Of twelve MSI-H cancers, six failed to express MLH1 and MSH2. Interestingly, ten cancers showed loss of expression of MLH1 and/or MSH2 protein. Of three cancers with Chk1 frameshift mutation, two failed to express MSH2 alone (Table 1). Chk1 frameshift mutation and BRAF mutation in colorectal cancers Enrichment and direct sequence analysis of the genomic DNA led to the identification of two types of Chk1 frameshift mutation in 5 colorectal cancers (Fig. 3). Two and three of these types were found in sporadic colorectal cancers and HNPCC, respectively. Interestingly, all the mutations were detected in cases with MSI-H. One type of Chk1 mutation was changed from poly(A)9 to poly(A)8 in the 93-base pair coding region encompassing the Chk1 poly(A)9 tracts and the other was changed to poly(A)10. The Chk1 mutations of poly(A)8 and poly(A)10 led to a reading frameshift and a premature stop codon at positions 238 and 243 amino acids, respectively, compared with the normal length of 476 amino acids. Pathologically, 2 Chk1 mutations were found in the cases with lymph node metastasis and clinical stage C. Three cases with Chk1 mutation were of clinical stage B without lymph node metastasis. Histologically, all the mutations were seen in moderately-differentiated adenocarcinoma. In addition, we searched for BRAF mutation in exon 11 and 15, which contain mutational hot spot, and no mutations were found in both sporadic colorectal cancers and HNPCCs. We repeated the experiment three times and found that the data were consistent. Discussion Since tumors with MSI accumulate hundreds or thousands of mutations in the mononucleotide repeats
Figure 2. Expression of MLH1 and MSH2 protein in HNPCCs. Normal colonic mucosa exhibited positive staining for the MSH2 protein (A). HNPCC displayed moderate to strong immunostaining for MSH2 in nucleus of cancer cells (B). Another HNPCC showed immunonegativity for the MSH2 proteins in cancer cells (C). (Original magnification, 100).
throughout the genome,3,29 the detection of insertion or deletion mutations in these mononucleotide repeats is indicative of widespread genomic damage with functional significance for cancer development. For example, cancers with MSI frequently contain frameshift mutations in a repetitive sequence within the coding region of genes, such as transforming growth factor receptor type II (TGFbRII ),
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Table 1 MSI and framshift mutation of hCHK1 detected in HNPCC & MLH-1 and MSH-2 protein immunoreactivity in the HNPCC patients Case
Poly A(9) alteration
Stop codon
BAT-26
BAT-25
BAT-34
D2S123
D17S250
APC II
MLH-1
MSH-2
1 2 3 4 5 6 7 8 9 10 11 12 13 14
e e e e e e e e Poly A(8) e Poly A(8) e Poly A(8) e
e e e e e e e e 238 e 238 e 238 e
þ þ þ þ þ þ þ þ þ þ þ þ
nd þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ
þ þ LOH þ þ nd þ þ þ þ þ þ þ
þ nd þ þ nd þ þ þ þ þ þ LOH
þ þ þ þ þ þ þ LOH þ þ þ þ þ
þ þ þ þ þ þ þ
þ þ þ þ þ þ þ
, MSI-negative; þ, MSI-positive; LOH, loss of heterozygosity; nd, not determined.
the insulin-like growth factor II receptor (IGFIIR), and the proapoptotic genes BAX and ICE.30e32 In the present study, we analyzed the presence of MSI and frameshift mutations of Chk1 containing a mononucleotide repeat and BRAF
Figure 3. Chk1 frameshift mutation in colorectal cancers. Cyclic sequencing analysis was performed using the genomic DNA of the case 13 and case 22 tumors. Case 13 showed a deletion of nucleotide A, which led to a reading frameshift mutation and a premature stop codon at amino acid 238, compared with normal poly(A)9. Case 22 showed an insertion of nucleotide A, which led to a reading frameshift mutation and a premature stop codon at amino acid 243. N, normal DNA; T, tumor DNA.
mutations in sporadic colorectal cancers and HNPCCs. Interestingly, MSI-positivity was found in 11 out of 51 sporadic cancers and in all of the HNPCCs. The Chk1 frameshift mutation was also detected in five cancers, two sporadic colorectal cancers and three HNPCC, and observed in 3 and 2 cases with clinical stage B and C, respectively. Interestingly, Chk1 frameshift mutations were found only in the cases with MSI-H, suggestive of the etiological role of the deficient DNA mismatch repair. The Chk1 mutations were rare event in colon cancer overall, but should be considered a common event in the MSI cancer cells. Thus, these results suggest that the Chk1 gene is a target of genomic instability in MSI colorectal tumorigenesis and that Chk1 frameshift mutations may be a consequence of mismatch repair deficiency in cancer cells. Cells are continually exposed to a wide variety of forces and toxins that are capable of damaging DNA. The human homolog of the checkpoint kinase, Chk1, becomes phosphorylated in a manner that is dependent on the activity of Atm and/or Atr. Chk1 phosphorylates at least three DNA damage-inducible phosphorylation sites in p53,33 which prevents Mdm2 binding resulting in p53 stabilization. An association between Chk1 with the 14-3-3 proteins is stimulated by the phosphorylation of Chk1, and a region of Chk1 within the non-catalytic domain, which includes amino acids 285e312, and appears to be important for interacting with the 14-3-3 proteins.34 The Chk1 mutations detected in this study led to a reading frameshift and a premature stop codon at the amino acids at positions 238 and 243, respectively. Although functional analysis of these mutant proteins was not performed in this study, our results suggest that frameshift mutations in poly(A)9 tracts of the Chk1 gene may cause a defect in response to DNA damage and mutational inactivation of the Chk1 gene is a specific event in colorectal cancers with MSI. Recently, it has been suggested that BRAF mutations in colorectal cancers is linked to the proficiency of repairing mismatch.35 Lubomierski et al. found that BRAF was mutated more often in colorectal cancers with MSI than
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without MSI. The most prevalent BRAF alteration, V599E, occurred only in tumors with MSI. However, BRAF mutations were absent in eastern HNPCCs,24 which is typical mismatch repair deficient cancers, and gastric cancers.36 In the present study, no BRAF mutations were detected in both sporadic colorectal cancer and HNPCC with MSI. Although our results may underestimate the prevalence of BRAF mutations in colorectal cancers, it is likely that BRAF mutations may not be linked with defective mismatch repair in eastern colorectal cancers. Additional studies with a larger patient cohort are needed to verify these initial observations.
Acknowledgment This work was supported by the Korea Science & Engineering Foundation (KOSEF) through the Cell Death Disease Research Center at The Catholic University of Korea.
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14. Wang XW, Harris CC. p53 tumor-suppressor gene: clues to molecular carcinogenesis. J Cell Physiol 1997;173:247–55. 15. Al-Khodairy F, Fotou E, Sheldrick KS, et al. Identification and characterization of new elements involved in checkpoint and feedback controls in fission yeast. Mol Biol Cell 1994;5:147–60. 16. Walworth N, Davey S, Beach D. Fission yeast chk1 protein kinase links the rad checkpoint pathway to cdc2. Nature 1993;363: 368–71. 17. Nakajo N, Oe T, Uto K, Sagata N. Involvement of Chk1 kinase in prophase I arrest of Xenopus oocytes. Dev Biol 1999;207:432–44. 18. Sanchez Y, Wong C, Thoma RS, et al. Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25. Science 1997;277:1497–501. 19. Wang JYJ. Cellular responses to DNA damage. Curr Opin Cell Biol 1998;10:240–7. 20. Bertoni F, Codegoni AM, Furlan D, et al. CHK1 frameshift mutations in genetically unstable colorectal and endometrial cancers. Genes Chromosomes Cancer 1999;26:176–80. 21. Davis H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature 2002;417:949–54. 22. Yuen ST, Davies H, Chan TL, et al. Similarity of the phenotypic patterns associated with BRAF and KRAS mutations in colorectal neoplasia. Cancer Res 2002;62:6451–5. 23. Lubomierski N, Plotz G, Wormek M, et al. BRAF mutations in colorectal carcinoma suggest two entities of microsatellite-unstable tumors. Cancer 2005;104:952–61. 24. Miyaki M, Iijima T, Yamaguchi T, et al. Both BRAF and KRAS mutations are rare in colorectal carcinomas from patients with hereditary nonpolyposis colorectal cancer. Cancer Lett 2004;211:105–9. 25. Astler VB, Coller FA. The prognostic significance of direct extension of carcinoma of the colon and rectum. Ann Surg 1954;139:846–51. 26. Vassen HFA, Watson P, Mecklin JP, Lynch HT. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC. Gastroenterology 1999;116:1453–6. 27. Lee JY, Dong SM, Kim SY, et al. A simple, precise and economical microdissection technique for analysis of genomic DNA from archival tissue sections. Virchows Arch 1998;433:305–9. 28. Pedroni M, Tamassia MG, Percesepe A, et al. Microsatellite instability in multiple colorectal cancer. Int J Cancer 1999;81:1–5. 29. Yamamoto H, Sawai H, Perucho M. Frameshift somatic mutations in gastrointestinal cancer of the microsatellite mutator phenotype. Cancer Res 1997;57:4420–6. 30. Markowitz SD, Wang J, Myeroff L, et al. Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. Science 1995;268:1336–8. 31. Rampino N, Yamamoto H, Ionov Y, et al. Somatic frameshift mutations in the BAX gene in colon cancers of the microsatellite mutator phenotype. Science 1997;275:967–9. 32. Souza RF, Appel R, Yin J, et al. Microsatellite instability in the insulin-like growth factor II receptor gene in gastrointestinal tumours. Nat Genet 1996;14:255–7. 33. Shieh SY, Ahn J, Tamai K, Taya Y, Prives C. The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. Genes Dev 2000;14:289–300. 34. Chen L, Liu TH, Walworth NC. Association of Chk1 with 14-3-3 proteins is stimulated by DNA damage. Gene Dev 1999;13:675–85. 35. Rajagopalan H, Bardelli A, Lengauer C, et al. Tumorigenesis: RAF/ RAS oncogenes and mismatch-repair status. Nature 2002;418:934. 36. Kim IJ, Park JH, Kang HC, et al. Mutational analysis of BRAF and K-ras in gastric cancers: absence of BRAF mutations in gastric cancers. Hum Genet 2003;114:118–20.
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Chk1 frameshift mutation in sporadic and hereditary non-polyposis colorectal cancers with microsatellite instability C.J. Kim a, J.H. Lee a, J.W. Song, Y.G. Cho, S.Y. Kim, S.W. Nam, N.J. Yoo, W.S. Park, J.Y. Lee* Department of Pathology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Republic of Korea Accepted 15 February 2007 Available online 3 April 2007
Abstract Aim: Protein kinase Chk1 (hChk1) is essential in human cells for cell cycle arrest in response to DNA damage, and has been shown to play an important role in the G2/M checkpoint. The BRAF mutations have been suggested to be linked with defective mismatch repair in colorectal cancers. The aim of this study was to investigate whether a frameshift mutation within the Chk1 gene contribute to the development or progression of eastern sporadic and hereditary non-polyposis colorectal cancer (HNPCC) with microsatellite instability (MSI). Methods: We analyzed MSI using the 6 microsatellite markers and a frameshift mutation in the BRAF gene and in poly(A)9 within the Chk1 gene in 51 sporadic colorectal cancer and 14 HNPCC specimens. Results: Eleven of the 51 sporadic colorectal cancers and all of the 14 HNPCCs were MSI-positive. Chk1 frameshift mutations were observed in 2 and 3 sporadic colon cancers and HNPCC, respectively, whereas no BRAF mutations were detected in these samples. Interestingly, all cases with the Chk1 frameshift mutation had high-frequency MSI. Conclusion: These results suggest that the Chk1 gene is a target of genomic instability in MSI-positive colorectal cancers and that the Chk1 framshift mutations might be involved in colorectal tumourigenesis through a defect in response to DNA damage in a subset of sporadic colorectal cancers and HNPCCs. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Protein kinase Chk1 (CHK1); Microsatellite instability (MSI); BRAF; Mutation; Colorectal cancers
Introduction Microsatellite repeats are widely distributed throughout the genome. The genes for mismatch repair are responsible for eliminating insertion or deletion mutations in these repeat sequences that arise during DNA replication. Inactivation of the genes responsible for mismatch repair induces a deficiency in mismatch repair, which leads to instability of the microsatellite sequence such as poly(A) or poly(CA) repeats and results in a mutator phenotype. Defective mismatch repair is believed to promote tumorigenesis by accelerating the accumulation of genetic alterations in oncogenes and/or tumor suppressor genes. Microsatellite instability (MSI) is a form of genetic instability in virtually * Corresponding author. Tel.: þ82 2 590 1190; fax: þ82 2 537 6586. E-mail address: [email protected] (J.Y. Lee). a Both these authors contributed equally to this work. 0748-7983/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ejso.2007.02.007
all tumors from patients with HNPCC and a subset of various sporadic tumors, including colorectal and gastric cancer.1e5 It was reported that more than 90% of colorectal cancers in HNPCC patients are MSI, whereas only 15% of sporadic colorectal cancers are MSI.6 However, molecular mechanism of MSI in colorectal carcinogenesis is still unclear. The cell has developed screening systems for maintaining the stability of genetic material. These systems detect DNA damage and facilitate its repair by imposing checkpoint arrests on the cell cycle.7 A major checkpoint of the cell cycle occurs at the end of the G1 phase and prior to entry into the S phase. A second major checkpoint exists at the exit from the G2 phase and before the entry into the M phase.8e10 Indeed, one of the hallmarks of cancer cells is a loss of a checkpoint controlling mechanism.11e14 The Chk1 gene, which is a homolog of the Schizosaccharomyces pombe Chk1 protein kinase,15,16 is an important component of the G2 checkpoint.
C.J. Kim et al. / EJSO 33 (2007) 580e585
This gene phosphorylates Cdc25 and prevents the dephosphorylation of Cdc2.9,10,17e19 In this state, the cell cannot undergo mitosis. Interestingly, it has been shown that Chk1 gene sequence contains a tract of nine adenines in its coding region. Moreover, frameshift mutations in this tract have been found in western colorectal and endometrial cancers with a high degree of MSI.20 However, CHK1 frameshift mutations have not been studied in eastern population. Recently, the activating mutations of the BRAF gene have been reported in various cancers, including colorectal cancers21,22 and alterations of BRAF have been implicated in the carcinogenesis of colorectal tumors with MSI.23 However, extremely low frequency of BRAF mutation in eastern HNPCCs has been reported.24 Thus, it is important to investigate whether the BRAF mutations is linked with mismatch repair deficient colorectal cancers in eastern country. This study searched for MSI and mutations of the Chk1 and BRAF genes in 51 Korean sporadic colorectal cancer and 17 HNPCC specimens to determine if a defective mismatch repair systems results in Chk1 and BRAF mutations in eastern patients with colorectal cancer. Materials and methods Tumour specimens A total of 51 formalin-fixed, paraffin-embedded sporadic colon cancers and 14 HNPCC specimens were obtained from the College of Medicine, The Catholic University of Korea. HNPCC cases were located in the right-side colon and sporadic cancers were located in the left-side colon. Tumor masses ranged from 13 to 70 mm on longest diameter, with a mean diameter of 42 mm. Tumor stage was classified according to the Dukes’ criteria.25 There were 8, 24, 30, 3 cases with stage A, B, C, and D, respectively. Histologically, the cancers consisted of 2 well-differentiated, 62 moderately-differentiated, and 1 poorly-differentiated adenocarcinoma. Informed consent was obtained according to the Declaration of Helsinki. The institutional review board of the Catholic University of Korea, College of Medicine, approved this study. Fourteen HNPCC patients fulfilled for the clinical criteria for HNPCC.26 None of the patients with sporadic colorectal cancer had a family history of the disease. The Hematoxylin & Eosin (H & E) stained histological sections from each case were reviewed. Two pathologists screened the histological sections and selected the areas of representative tumor cells. Malignant cells were selectively procured from the H & E stained slides without contamination from normal cells using a laser microdissection device (ION LMD, JungWoo international Co, Seoul, Korea). The normal cells were also obtained from the corresponding cancer-free colonic mucosa. DNA extraction was carried out using a slight modification of single-step DNA extraction method previously described.27
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Immunohistochemistry for the MLH-1 and MSH-2 in HNPCC specimens MLH1 and MSH2 protein expression in HNPCC specimens was measured by immunohistochemistry. Two mm cancer tissue sections were cut the day before use and stained according to standard protocols. To maximize the signal on immunohistochemistry, two strategies were used in the present study, i.e. antigen retrieval in citrate buffer, and signal amplification with biotinylated tyramide. For the former, heat-induced epitope retrieval was conducted by immersing the slides in Coplin jars filled with 10 mmol/L citrate buffer (pH 6.0) and boiling the buffer for 30 min in a pressure cooker (Nordic Ware, Minneapolis, MN, USA) inside a microwave oven at 700 W; the jars were then cooled for 20 min. For the latter, the Renaissance TSA indirect kit (NEN Life Science, Boston, MA, USA), which included streptavidin-peroxidase and biotinylated tyramide, was used. After rinsing with PBS, the slides were treated with 1% H2O2 in PBS for 15 min at room temperature to abolish endogenous peroxidase activity. After washing with TNT buffer (0.1 mol/L TriseHCl, pH 7.4, 0.15 mol/L NaCl and 0.05% Tween 20) for 20 min, the slides were treated with TNB buffer (0.1 mol/L TriseHCl, pH 7.4, 0.15 mol/L NaCl and 0.5% blocking reagent). Sections were incubated overnight at 4 C with the antibody for MLH1 protein (EMD Biosciences, San Diego, CA, USA; 1:100) and MSH2 (EMD Biosciences, San Diego, CA, USA; 1:100). Detection was carried out using biotinylated goat anti-mouse antibody (Sigma, St. Louis, MO, USA), followed by incubation with peroxidase-linked avidin-biotin complex. Diaminobenzidine was used as chromogen, and the slide was counterstained with Mayer’s hematoxylin. The specificity of antibodies was confirmed in 9 cancer cell lines by Western blot analysis (data not shown). The tumors were interpreted as negative when immunostaining was weak, like the corresponding normal colonic mucosa, or when immunopositive cells were less than 30% of the cancer cells. The results were reviewed independently by 2 pathologists. As negative controls, the slide was treated by replacement of primary antibody with non-immune serum. MSI analysis Somatic microsatellite alterations were analyzed by polymerase chain reaction (PCR), as previously reported.28 In order to identify the MSI in cancer, the microsatellite markers, including BAT-25, BAT-26, BAT-34, D2S123, D17S250, and APCII, were recommended by a National Cancer Institute workshop.2 Only those cases showing an unequivocally distinct additional band or shifts in the tumor tissue DNA compared with normal tissue DNA were recorded and classified as MSI. According to the Bethesda criteria, tumors with MSI-positive cases in more than two informative markers were considered to be MSI-H.2
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Chk1 frameshift mutation and BRAF mutation analysis The frameshift mutation in the mononucleotides runs of the Chk1 gene were determined by amplifying genomic DNAs from each cancer cells and corresponding non-cancerous colonic mucosal tissues with the primer set covering the 93-base pair coding region encompassing the Chk1 poly(A)9 tracts. The primer sequence is as follows; 50 CTCGCTGGAGAATTGCCA-30 (sense) and 50 -TTTCCAA GGGTTGAGGTA-30 (antisense). Mutations in exon 11 and 15 of the BRAF gene, which contain mutational hot spots, were also examined. The primer sequences for exon 11 and 15 are as follows; for exon 11, 50 -GGACTCGAGT GATGATTGG-30 (sense) and 50 -GTGGTGACATTGTGA CAAGTC-30 (antisense) and for exon 15, 50 -TTCCT TTACTTACTACACCTCA-30 (sense) and 50 -GTTGTCTG GATCCATTTTGT-30 . Each polymerase chain reaction (PCR) procedure was performed under standard conditions in a 10 ml reaction mixture containing 1 ml of template DNA, 0.5 mM of each primer, 0.2 mM of each deoxynucleotide triphosphate, 1.5 mM MgCl2, 0.4 unit of Ampli Taq
gold polymerase (Perkin-Elmer, Foster City, CA), 0.5 mCi of [32P]dCTP (Amersham, Buckinghamshire, UK), and 1 ml of 10 buffer. The reaction mixture was denatured for 12 min at 94 C and incubated for 35 cycles (denaturing for 40 s at 94 C, annealing for 40 s at 50e56 C and extension for 40 s at 72 C). A final extension step was carried out for 5 min at 72 C. After amplification, the PCR products were denatured for 5 min at 95 C in a 1:1 dilution of a sample buffer containing 98% formamide/5 mmol/L NaOH. The products were loaded onto a single SSCP gel (FMC Mutation Detection Enhancement system; Intermountain Scientific, Kaysville, UT) with 10% glycerol. After electrophoresis, the gels were transferred to 3 MM Whatman paper and dried. Autoradiography was then performed using Kodak X-OMAT film (Eastman Kodak, Rochester, NY). When a band shift was observed in a sample, it was verified by repeating the SSCP procedure including PCR and subjecting the PCR products to DNA sequencing. All mutations were verified through triplicate experiments including tissue microdissection, PCR, SSCP, and direct sequencing analysis to ensure the specificity of the results.
Figure 1. Autoradiograms of MSI analysis in HNPCC case No. 13 are shown for each of the 6 MSI markers. The case shows MSI at all 6 loci (BAT-26, BAT-25, BAT-34, D2S123, D17S250, and APCII ). The arrowheads indicate MSI in the tumor DNA. N, normal DNA; T, tumor DNA.
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Results MSI in colorectal cancers MSI-positive cases were found in 11 out of 51 sporadic colorectal cancers and all of the HNPCCs, respectively. Interestingly, 12 of the HNPCC cases showed widespread high frequency MSI (MSI-H), whereas only 4 sporadic colon cancers showed MSI-H. Considering the pattern of MSI, all high frequency MSI cases tended to be MSI-positive in the BAT series. Fig. 1 gives representative examples of MSI. Immunohistochemistry in HNPCCs Normal colonic mucosa showed moderate to strong expression of MLH1 and MSH2 proteins mainly in the nucleus of the glandular epithelial cells. Expression of MLH1 and MSH2 in tumor cells was compared with their corresponding normal colonic mucosa. Finally, loss of expression of MLH1 and MSH2 proteins was detected in 7 (50%) of 14 HNPCCs (Fig. 2). Of twelve MSI-H cancers, six failed to express MLH1 and MSH2. Interestingly, ten cancers showed loss of expression of MLH1 and/or MSH2 protein. Of three cancers with Chk1 frameshift mutation, two failed to express MSH2 alone (Table 1). Chk1 frameshift mutation and BRAF mutation in colorectal cancers Enrichment and direct sequence analysis of the genomic DNA led to the identification of two types of Chk1 frameshift mutation in 5 colorectal cancers (Fig. 3). Two and three of these types were found in sporadic colorectal cancers and HNPCC, respectively. Interestingly, all the mutations were detected in cases with MSI-H. One type of Chk1 mutation was changed from poly(A)9 to poly(A)8 in the 93-base pair coding region encompassing the Chk1 poly(A)9 tracts and the other was changed to poly(A)10. The Chk1 mutations of poly(A)8 and poly(A)10 led to a reading frameshift and a premature stop codon at positions 238 and 243 amino acids, respectively, compared with the normal length of 476 amino acids. Pathologically, 2 Chk1 mutations were found in the cases with lymph node metastasis and clinical stage C. Three cases with Chk1 mutation were of clinical stage B without lymph node metastasis. Histologically, all the mutations were seen in moderately-differentiated adenocarcinoma. In addition, we searched for BRAF mutation in exon 11 and 15, which contain mutational hot spot, and no mutations were found in both sporadic colorectal cancers and HNPCCs. We repeated the experiment three times and found that the data were consistent. Discussion Since tumors with MSI accumulate hundreds or thousands of mutations in the mononucleotide repeats
Figure 2. Expression of MLH1 and MSH2 protein in HNPCCs. Normal colonic mucosa exhibited positive staining for the MSH2 protein (A). HNPCC displayed moderate to strong immunostaining for MSH2 in nucleus of cancer cells (B). Another HNPCC showed immunonegativity for the MSH2 proteins in cancer cells (C). (Original magnification, 100).
throughout the genome,3,29 the detection of insertion or deletion mutations in these mononucleotide repeats is indicative of widespread genomic damage with functional significance for cancer development. For example, cancers with MSI frequently contain frameshift mutations in a repetitive sequence within the coding region of genes, such as transforming growth factor receptor type II (TGFbRII ),
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Table 1 MSI and framshift mutation of hCHK1 detected in HNPCC & MLH-1 and MSH-2 protein immunoreactivity in the HNPCC patients Case
Poly A(9) alteration
Stop codon
BAT-26
BAT-25
BAT-34
D2S123
D17S250
APC II
MLH-1
MSH-2
1 2 3 4 5 6 7 8 9 10 11 12 13 14
e e e e e e e e Poly A(8) e Poly A(8) e Poly A(8) e
e e e e e e e e 238 e 238 e 238 e
þ þ þ þ þ þ þ þ þ þ þ þ
nd þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ
þ þ LOH þ þ nd þ þ þ þ þ þ þ
þ nd þ þ nd þ þ þ þ þ þ LOH
þ þ þ þ þ þ þ LOH þ þ þ þ þ
þ þ þ þ þ þ þ
þ þ þ þ þ þ þ
, MSI-negative; þ, MSI-positive; LOH, loss of heterozygosity; nd, not determined.
the insulin-like growth factor II receptor (IGFIIR), and the proapoptotic genes BAX and ICE.30e32 In the present study, we analyzed the presence of MSI and frameshift mutations of Chk1 containing a mononucleotide repeat and BRAF
Figure 3. Chk1 frameshift mutation in colorectal cancers. Cyclic sequencing analysis was performed using the genomic DNA of the case 13 and case 22 tumors. Case 13 showed a deletion of nucleotide A, which led to a reading frameshift mutation and a premature stop codon at amino acid 238, compared with normal poly(A)9. Case 22 showed an insertion of nucleotide A, which led to a reading frameshift mutation and a premature stop codon at amino acid 243. N, normal DNA; T, tumor DNA.
mutations in sporadic colorectal cancers and HNPCCs. Interestingly, MSI-positivity was found in 11 out of 51 sporadic cancers and in all of the HNPCCs. The Chk1 frameshift mutation was also detected in five cancers, two sporadic colorectal cancers and three HNPCC, and observed in 3 and 2 cases with clinical stage B and C, respectively. Interestingly, Chk1 frameshift mutations were found only in the cases with MSI-H, suggestive of the etiological role of the deficient DNA mismatch repair. The Chk1 mutations were rare event in colon cancer overall, but should be considered a common event in the MSI cancer cells. Thus, these results suggest that the Chk1 gene is a target of genomic instability in MSI colorectal tumorigenesis and that Chk1 frameshift mutations may be a consequence of mismatch repair deficiency in cancer cells. Cells are continually exposed to a wide variety of forces and toxins that are capable of damaging DNA. The human homolog of the checkpoint kinase, Chk1, becomes phosphorylated in a manner that is dependent on the activity of Atm and/or Atr. Chk1 phosphorylates at least three DNA damage-inducible phosphorylation sites in p53,33 which prevents Mdm2 binding resulting in p53 stabilization. An association between Chk1 with the 14-3-3 proteins is stimulated by the phosphorylation of Chk1, and a region of Chk1 within the non-catalytic domain, which includes amino acids 285e312, and appears to be important for interacting with the 14-3-3 proteins.34 The Chk1 mutations detected in this study led to a reading frameshift and a premature stop codon at the amino acids at positions 238 and 243, respectively. Although functional analysis of these mutant proteins was not performed in this study, our results suggest that frameshift mutations in poly(A)9 tracts of the Chk1 gene may cause a defect in response to DNA damage and mutational inactivation of the Chk1 gene is a specific event in colorectal cancers with MSI. Recently, it has been suggested that BRAF mutations in colorectal cancers is linked to the proficiency of repairing mismatch.35 Lubomierski et al. found that BRAF was mutated more often in colorectal cancers with MSI than
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without MSI. The most prevalent BRAF alteration, V599E, occurred only in tumors with MSI. However, BRAF mutations were absent in eastern HNPCCs,24 which is typical mismatch repair deficient cancers, and gastric cancers.36 In the present study, no BRAF mutations were detected in both sporadic colorectal cancer and HNPCC with MSI. Although our results may underestimate the prevalence of BRAF mutations in colorectal cancers, it is likely that BRAF mutations may not be linked with defective mismatch repair in eastern colorectal cancers. Additional studies with a larger patient cohort are needed to verify these initial observations.
Acknowledgment This work was supported by the Korea Science & Engineering Foundation (KOSEF) through the Cell Death Disease Research Center at The Catholic University of Korea.
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