1 Division of Cell Biology, Floor 9, Department of Biomedicine and Surgery, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden, 2 Department of Natural Science and Biomedicine, University College of Health Sciences, Jönköping, Sweden and 3 Molecular Biology Laboratory, Strategic Development-LMÖ/IBK, University Hospital, Linköping, Sweden
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Abstract |
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Abbreviations: APC, adenomatous polyposis coli; CRC, colorectal cancer; DCC, deleted in colorectal cancer; ERK-1/-2, extracellular regulated kinases; MAPK, mitogen-activated protein kinase; MAPKKs, MAP kinase kinases; MAPKKKs, MAP kinase kinase kinases; MCR, mutation cluster region; MDE, mutation detection enhancement; MEK-1/-2, Map or Erk kinases; MSI, microsatellite instability; SSCA, single strand conformation analysis
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Introduction |
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Materials and methods |
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The age of the patients ranged from 25 to 93 years (median age 74 years). The stage of the tumor was described according to Duke's staging system: 17 of the tumors were in Duke's stage A, 58 in B, 42 in C and 7 in D. Five adenomas were also included in the study. 73 tumors were located in the colon (caecum 12; ascending colon 24; transverse colon 4; descending colon 2; sigmoid colon 31) and 55 in the rectum. Seventy-six of the tumors displayed an ulcerative phenotype, 36 were polypoid and 5 showed a mixed ulcerative and polypoid phenotype (13 of the tumors were not classified). Smoking data was available for 51 of the patients: 25 were smokers and 26 non-smokers. The study protocol was approved by the research ethics committee at the Faculty of Health Sciences, Linköping, Sweden (no. 98113).
RNA extraction and RTPCR
RNA was extracted with a RNeasy® Mini Kit (Qiagen, Hilden, Germany) according to the supplier's recommendations from 1530 mg intestinal tumor tissue from three different patients. RNA was also extracted from the corresponding normal tissue from the same patient, surgically resected 10 cm from the tumor. The RNA was on-column DNase treated with a RNase-free DNase Set (Qiagen) according to the manufacturer's protocol. Equal amounts of total RNA were used for cDNA synthesis, which was performed with Superscript IITM RT (Invitrogen, Stockholm, Sweden) and random primers (Invitrogen) with RNase-free utilities. The samples were stored at -70°C until used.
Aliquots of 1 µl of tumor or corresponding normal cDNA were amplified in 20 µl multiplex PCR reactions containing 75 mM TrisHCl (pH 9.0), 20 mM (NH4)2SO4, 0.01% Tween 20, 2 mM MgCl2, 200 µM each dGTP, dATP, dTTP and dCTP, 1 µM each ARAF, BRAF or RAF-1 primer and 0.1 µM each housekeeping gene primer (Table I), 0.5 U Taq DNA polymerase (Invitrogen). The PCR temperature profile was 1 cycle of 94°C for 2.5 min, followed by 35 cycles of 94°C for 1 min, annealing temperature for 1 min (Table I) and 1 min at 72°C, with final extension at 72°C for 7 min. The primers targeted to regions with as little homology as possible between the three RAF genes and at least one of the primers in each set should cover exon/exon boundaries. After amplification, the PCR products were separated on 1.5% agarose gels, stained with ethidium bromide and visualized in UV light.
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PCR amplification of genomic DNA
Aliquots of 20 ng of genomic tumor or corresponding normal genomic DNA were amplified in 20 µl PCR reactions as described above (except for the housekeeping gene). Intronic primers were used for amplification of the ARAF, BRAF, RAF-1, KRAS and p53 genes, covering exons 10 and 13 of the ARAF gene, 11 and 15 of the BRAF gene, 10 and 14 of the RAF-1 gene, 1 and 2 of the KRAS gene and 58 of the p53 gene (Table I). Exonic primers were used to amplify the hot-spot codons in exon 3 of the ß-catenin gene and the overlapping PCR fragments of the mutation cluster region (MCR) in exon 15 (codons 832892 and 12421584) of the APC gene. The PCR temperature profile and agarose gel detection was performed as described above.
Mutation analysis
An aliquot of 1 µl of the primary PCR product was reamplified for single strand confirmation analysis (SSCA) in a similar reaction mixture to that described above, with the addition of 1 µCi [32P]dATP per sample (3000 Ci/mmol; Amersham Biosciences, Piscataway, NJ) for 8 cycles. Samples of 2 µl of the labeled PCR products were diluted with 6 µl of 0.2% SDS, 20 mM EDTA and 12 µl of loading buffer (98% formamide, 9.8 mM EDTA, pH 8.0, 0.098% bromphenol blue and xylene cyanol FF), heated to 95°C for 5 min and applied to a non-denaturing 6% polyacrylamide gel containing 10% glycerol and a 0.5x MDETM gel (Mutation Detection Enhancement; BMA Products, Rockland, ME). Electrophoresis was performed at 46 W constant power at room temperature for 1217 h. The gels were transferred to a 3MM filter paper and exposed to X-ray film at -70°C for 417 h with intensifying screens. Shifted fragments were cut out from the gel, eluted in water, reamplified and purified with GFXTM PCR DNA and a Gel Band Purification Kit (Amersham Biosciences) for DNA sequencing analysis. All mutations identified were confirmed by a second mutational analysis of the original DNA sample.
DNA sequencing
Mobility shifted, reamplified and purified samples were sequenced with a ThermoSequenase Radioabeled Termination Cycle Sequencing Kit (USB Corp., Cleveland, OH) and 33P-labeled ddNTPs (Amersham Biosciences) according to the supplier's protocol. The samples were diluted with 4 µl of denaturing loading buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol FF) and electroporesed in 6% denaturing polyacrylamide gels containing 7 M urea for 12 h at 70 W and 50°C. The gels were dried and autoradiography was performed at room temperature for 4872 h.
MSI analysis
MSI analysis was performed in a similar fashion to that described in Jansson et al. (13). In brief, 20 ng of tumor and paired normal DNA from each patient was amplified with primers for the microsatellite marker BAT-26 (14), for 35 cycles at 57°C annealing temperature, and labeled with 1 µCi [32P]dATP per sample for 8 cycles as described above. The fragments were mixed with denaturing loading buffer and separated on a sequencing gel (see above), transferred to a filter paper and autoradiography was performed. MSI was visually estimated by two independent observers and MSI was confirmed in a separate experiment.
Statistical analysis
Fishers exact test, the MannWhitney U-test and 2 analysis were performed with the SPSS 11.0.0 statistical software package (SPSS Inc., Chicago, IL). The results were considered significant at the P < 0.05 level.
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Results |
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Mutation screening of the MAPK pathway
Mutation analysis was performed for the known hot-spot regions of the BRAF and KRAS genes in 130 primary CRC tumors. The results were correlated with clinical and mutational data of the APC/ß-catenin and p53 pathways (Tables II and III) and MSI (Table II). We also studied the corresponding regions of ARAF and RAF-1, similar to exons 11 and 15 in the BRAF gene (Table IV). SSCA combined with direct DNA sequencing analysis revealed somatic mutations in the MAPK pathway in 50.7% of the primary CRC tumors. The KRAS and BRAF genes exhibited missense mutations in 52/130 (40%) and 15/130 (11.5%) of the tumors, respectively.
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As expected, a high frequency of mutations was found in the KRAS gene (Table III). The majority of the mutations were located in exon 1 and only one mutation was found in exon 2. Codon 12 harbored the highest number of genetic alterations with a frequency of 43/52 (83%). Codons 13 and 61 displayed mutations in 8/52 (15%) and 1/52 (2%), respectively. The most frequent nucleotide substitution was a G T transversion that changed glycine to valine and occurred in 14/43 (32%) of the KRAS mutated tumors.
BRAF/KRAS mutations and MSI
In total, 12/130 (9%) of all the tumors were microsatellite unstable for the BAT-26 marker. Six of 15 tumors (40%) with BRAF mutations were microsatellite unstable, compared with six of 115 (5%) tumors without BRAF mutations displaying MSI (P < 0.001, Pearson 2 test). All of the six microsatellite unstable tumors with BRAF mutations displayed the V599E mutation and no MSI was found in tumors with BRAF mutations in the other codons.
Consequently, a significantly low incidence of MSI in KRAS mutated tumors was also observed (P = 0.027, Fisher's exact test) and in only one single case out of 52 KRAS mutations was MSI found. The KRAS mutation in this microsatellite unstable tumor was found in codon 12, yielding a G D amino acid shift.
BRAF/KRAS mutations and correlation to clinical data
The BRAF mutations were also subjected to statistical analysis with respect to available clinical data for CRC tumors. The majority of the BRAF mutated tumors were located in the colon (87%) rather than the rectum (13%, P = 0.014, Fisher's exact test). However, no such correlation between tumor localization and KRAS mutations was evident (P = 0.45, Pearson 2). Eight BRAF mutated tumors arose from the right side of the colon and three from the left side, but no significant difference could be found (P = 0.137, Pearson
2). Previously, an association was reported between mutations in codon 12 of the KRAS gene and polypoid tumor growth in CRC (15), but no such correlation could be observed in our study. Furthermore, no correlation with other clinico-pathological features could be found between mutations in KRAS or BRAF and age, gender, Duke's stage or smoking data (MannWhitney U-test or
2 test).
Mutation screening of the APC, ß-catenin and p53 genes
Mutation screening of the APC and ß-catenin genes revealed mutations in 38/130 (29%) and 1/130 (0.8%) CRC tumors, respectively. The majority of the APC mutations were deletions or insertions yielding a truncated protein. Mutations in the p53 gene were found in 54/130 (42%) of the tumors and the majority of the mutations were missense mutations. Detailed results from the mutation screening of the APC, ß-catenin and p53 genes are available upon request.
Correlation between genetic alterations
A negative correlation between mutations in two cancer genes may reflect a similar mechanism for tumor-specific activation of that pathway. In agreement with earlier studies (8,9), statistical analysis showed that mutations in the KRAS and BRAF genes were mutually exclusive and in no case in the present study could such mutations be observed simultaneously.
Furthermore, we analyzed the BRAF/KRAS mutation data in relation to mutations in the p53 and APC/ß-catenin pathways. Ten of fifteen (60%) of the tumors with BRAF mutations did not display mutations in any of the investigated hot-spot regions of the APC/ß-catenin or p53 genes. The corresponding number of tumors with KRAS mutations without mutations in these pathways was 20/52 (38.5%). In total, 29/130 (22.3%) tumors did not display mutations in either of the genes investigated in the MAPK, APC/ß-catenin or p53 pathways, which suggests tumorigenic activation through other mechanisms or genes than those studied in the present investigation. No significant patterns of KRAS and/or BRAF mutations related to genetic alterations in the APC/ß-catenin and p53 genes were evident (Fisher's exact test and 2 test). The APC/ß-catenin/p53 mutational data were also tested for correlations with each other, but no significant results were found, which indicates more complex regulatory mechanisms in the large network of genetic alterations in these pathways.
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Discussion |
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In the present study all of the mutations found were located in exon 15 of the BRAF gene, between codons 593 and 600. All of the mutations found in the BRAF gene were somatic and were not found in corresponding normal leukocyte DNA, which supports an active role for these BRAF mutations in tumor progression. In good agreement with Rajagopalan et al. (9) and Yuen et al. (10), 83% of the mutations were the T A transversion found in codon 599, which indicates a crucial role for this amino acid in BRAF gene activation. Several of the mutations identified by Davies et al. (8) displayed an increased kinase activity in vitro and the V599E mutated variant showed a 10-fold increased activity compared with wild-type BRAF activity in COS cells (8). The amino acid valine has been conserved throughout evolution and is located close to the conserved T598 and S601, which are required for the kinase activity of BRAF (16). These observations might be indicative of the importance of this amino acid. We also found one novel and one previously identified (10) mutation in exon 15. Codon 600 has earlier been reported to display the K600E mutation (9). In the present study we found K600N and D593G mutations in the BRAF gene, which both are located in an evolutionarily conserved region of the RAF genes (8).
Furthermore, mutations in BRAF exon 11 were earlier identified in only 1/33 and 3/333 CRC tumors, xenografts or cell lines (8,9). Previously, Yuen et al. (10) found 2/215 exon 11 mutants in colorectal tumors. In all cancer types in total, only a limited frequency of exon 11 mutations have been reported so far in human tumors (810,17,18), targeting either the phosphorylation sites for Akt or the glycine-rich domain, responsible for orientation of the ATP molecule (8). Exon 11 mutations therefore seem to be less frequent than exon 15 mutations, which may indicate a different activating mechanism for mutations in exon 11, engaging a different intracellular signaling pathway.
Mutations in the KRAS gene were found in 40% of the tumors in codons 12, 13 and 61, which are responsible for the transforming potential of the KRAS oncogene (19), which is consistent with earlier reports (20,21). The ras gene products are located upstream of the three RAF genes in the MAPK signaling cascade and binds to the conserved region I in the BRAF and RAF-1 gene products (22). After activation of the BRAF and RAF-1 gene products, the conserved regions II and III are needed for interaction with the downstream MEK-1 and MEK-2 proteins (22). BRAF is the strongest activator of MEK-1 and MEK-2 compared with RAF-1 (23). The role of ARAF in activating MEK-1 and MEK-2 seems to be of limited importance (24), which is also supported by the present study, in which mutational activation of ARAF could not be seen. In agreement with Davies et al. (8) and Rajagopalan et al. (9), none of the KRAS mutated tumors contained mutations in the BRAF gene or vice versa. Papin et al. (22) showed that KRAS and BRAF are located in the same regulatory pathway and that a mutation in either gene gives a selective proliferative advantage.
We also found a special preference for BRAF mutations in the colonic part compared with rectal tumors. Interestingly, an earlier study (20) found more KRAS mutations in rectal compared with colonic tumors. However, this distribution pattern could not be confirmed in our study. Yuen et al. (10) found a higher preference for BRAF mutations in the earlier Duke's tumor stages. However, this trend is neither supported by us nor by the study made by Rajagopalan et al. (9). Yuen et al. (10) also found a slight, but non-significant, preference for BRAF mutated tumors from the left colon, but we could see an opposite, but non-significant, pattern in our study.
In the present study we also found a significant association between BRAF mutations and MSI analyzed using the BAT-26 marker, which has been shown to detect nearly 100% of microsatellite unstable tumors (14,25). Our results therefore support the study made by Rajagopalan et al. (9), who also observed a correlation between BRAF mutations and MSI. However, Yuen et al. (10) could not confirm these results. A significantly lower incidence of MSI in KRAS mutated tumors was consequently found in the present study, which was earlier also shown by Jass et al. (26). These differences between the present study and the investigation made by Yuen et al. (10) may therefore depend on different lifestyle factors between Asian and Caucasian populations, such as diet, alcohol consumption and smoking.
We also tried to correlate mutations in the MAPK pathway with other genetic alterations in CRC, p53, APC and ß-catenin mutations, but no significant correlations could be found. However, the frequency of APC and ß-catenin mutations was lower than expected from other reports (27,28). This may partly be due to the analysis strategy, where the MCR of the APC gene and the GSK3ß phosphorylation sites of ß-catenin only were screened for mutations. However, according to the APC mutation database, this region covers the majority of APC mutations found in sporadic CRC. Twenty-two percent of the tumors did not reveal any mutation in the genes included in this study, which suggests that other genes of the studied pathways or other pathways and genes than those indicated in the KinzlerVogelstein model (2) of CRC may be involved.
Collectively, these results therefore imply increased importance of the MAPK pathway in CRC development, especially via mutational activation of the KRAS and BRAF genes.
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Note added in proof |
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Notes |
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Acknowledgments |
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References |
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