- Email: [email protected]
Mutational analysis of CASP1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 14 genes in gastrointestinal stromal tumors
Human Pathology (2009) 40, 868–871
www.elsevier.com/locate/humpath
Original contribution
Mutational analysis of CASP1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 14 genes in gastrointestinal stromal tumors☆ Yoo Ri Kim BS a , Kyoung Mee Kim MD b , Nam Jin Yoo MD a , Sug Hyung Lee MD a,⁎ a
Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Korea
b
Received 9 September 2008; revised 1 November 2008; accepted 7 November 2008
Keywords: Caspase; Mutation; GIST; Cancer; Apoptosis
Summary Deregulation of apoptosis is one of the hallmarks of cancer, and inactivation of cancer cell apoptosis has been reported in many cancers. Caspases, the main executioners during apoptosis and inflammation, have been reported to harbor inactivating mutations in several cancers. The aim of this study was to explore whether CASP1 to 10 and 14 genes that encode caspase 1 to 10 and 14 are somatically mutated in gastrointestinal stromal tumor. We analyzed the entire coding region and all splice sites of all 11 human CASP genes for the detection of somatic mutations in 22 gastrointestinal stromal tumors by a single strand conformation polymorphism assay. We found a recurrent CASP4 mutation (c.1093CNG [p.L365V]) in 4 gastrointestinal stromal tumors, but there were no mutations in the other 10 CASPs. The CASP4 mutation was a missense mutation and was predicted to substitute amino acids in the small protease subunit of caspase 4. Overall, the gastrointestinal stromal tumor tissues harbored a CASP mutation in 18.2% (4/22). Our data indicate that somatic mutation of the CASP4 gene is common in gastrointestinal stromal tumor and suggest a possibility that CASP4 mutation might lead to alteration of apoptotic or inflammatory function and contribute to the pathogenesis of some gastrointestinal stromal tumors. © 2009 Elsevier Inc. All rights reserved.
1. Introduction Apoptosis regulates normal tissue homeostasis, cellular differentiation, and development [1,2]. Apoptosis occurs through activation of a family of cysteine proteases that cleave the substrates at aspartate residues, known as caspases. The caspase family consists of 13 mammalian isoenzymes, of which 11 human isoenzymes are currently
☆ This work was supported by a grant from Korea Research Foundation in Korea (2007-314-E00042). ⁎ Corresponding author. E-mail address: [email protected] (S. H. Lee).
0046-8177/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2008.11.013
known [3,4]. The principal role of caspases is initiation and execution of apoptosis [3-4]. Caspases are classified as either effector or initiator caspases. The initiator caspases appear to be caspases 2, 8, 9, and 10, and the effector caspases appear to be caspases 3, 6, and 7 [3-4]. In addition, the interleukin 1β converting enzyme (caspase 1) subfamily caspases play a crucial role in both apoptosis and inflammation [3-8]. Caspase 14 is a unique caspase involved in cell differentiation and is not categorized into inflammatory or apoptotic caspases [9]. Inactivation of apoptosis allows cells to survive that are prone to genetic damage. Inactivation of apoptosis is one of the hallmarks of cancer [10]. Proapoptotic proteins are likely to be tumor suppressors, and inactivation of apoptosis by
Caspase mutation in GIST
869
somatic mutations has been reported in human cancers. In the caspase genes, somatic mutations of CASP3, CASP5, CASP7, CASP8, and CASP10 have frequently been detected in human cancer tissues [11-15], whereas those of CASP1, CASP4, CASP6, CASP9, and CASP14 have rarely been found [12,16-18]. Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors that are thought originate from the intestinal cells of Cajal [19]. GISTs are characterized by the expression of cKIT, a receptor tyrosine kinase. About 80% to 85% of GISTs harbor activating mutations of KIT or the platelet-derived growth factor receptor-α (PDGFRA) gene, which is considered to be the main mechanisms of GIST tumorigenesis [19]. Although most GISTs are responsive to imatinib (Gleevec) treatment, approximately 10% to 15% of GISTs show no response to imatinib (intrinsic or primary tumor cell resistance) [19]. Furthermore, GISTs do not respond to conventional chemotherapy considered standard for soft tissue sarcomas [19]. Together, these data suggest that GISTs might possess intrinsic mechanisms for apoptosis evasion. Also, alterations of inflammatory caspases are not known in GISTs. In this study, we analyzed 22 GISTs for the detection of somatic mutations in all types of caspase-encoding genes (CASP1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 14) that might contribute to the tumorigenesis of GISTs.
2. Materials and methods 2.1. Tissues and microdissection GIST tissues were obtained from 22 patients by surgical removal. Malignant and normal cells were selectively procured from hematoxylin and eosin-stained frozen sections using a 30G1/2 hypodermic needle (Becton Dickinson, Franklin Lakes, NJ) affixed to a micromanipulator by microdissection, as described previously [13-15]. DNA extraction was performed using a modified single-step DNA extraction method by proteinase K treatment, as described previously [13-15]. All of the patients were Asians
Table 1 Gene
(Korean). Approval was obtained from the institutional review board of College of Medicine, Catholic University of Korea, Seoul, Korea, for this study. The GISTs originated from stomach (n = 18), small intestine (n = 3), and esophagus (n = 1). Thirteen GISTs were less than 5 cm in diameter, and 9 were 5 to 10 cm in diameter. Fourteen GISTs showed less than 5 mitotic figures per 50 high-power fields, and 8 showed 5 to 10 mitotic figures per 50 high-power fields. The GISTs showed KIT mutations in 20 cases (20/22 [90.9%]). All of the KIT mutations were detected on exon 11, and they consisted of 11 deletions, 2 insertions, and 7 point mutations. Two GISTs showed neither KIT nor PDGFRA mutation. There were 4 high-risk, 7 intermediate-risk, and 11 low-risk GISTs.
2.2. Single strand conformation polymorphism analysis Genomic DNA from tumor cells and normal cells of corresponding patients was amplified with primer pairs covering the entire coding region of each CASP gene as described previously [11-18]. In the case of CASP1, we did not analyze exons 1 and 2 because the DNA sequence of CASP1 is exactly the same as that of the COP-1 gene [20]. Radioisotope was incorporated into the polymerase chain reaction (PCR) reactions for detection by autoradiogram. PCR and single strand conformation polymorphism (SSCP) analysis were performed as described previously [11-18]. After SSCP, DNA samples showing mobility shifts were cut from the dried gel and reamplified for 30 cycles using the same primer sets. DNA sequencing of the PCR products was carried out using a capillary automatic sequencer (ABI Prism Genetic Analyzer; Applied Biosystem, Foster City, CA) according to the manufacturer's recommendation.
2.3. Mutational analysis of KIT and PDGFRA Genomic DNA from the 22 GISTs was amplified by PCR with primer pairs covering exons 9, 11, 13, and 17 of the KIT gene and exons 12, 14, and 18 of the PDGFRA gene. Each
Summary of the CASP4 mutations in the GISTs Nucleotide change Exon (domain) (predicted amino acid change)
CASP4 c.1093CNG (p.L365V) CASP4 c.1093CNG (p.L365V) CASP4 c.1093CNG (p.L365V) CASP4 c.1093CNG (p.L365V) Abbreviation: HPF, high-power field.
8 (small protease 8 (small protease 8 (small protease 8 (small protease
Risk group
Tumor site
Size of Mitosis/50 KIT or PDGFRA tumor (cm) HPF mutation
Low
Stomach
5.0
0
p.Q575_R586dup
Low
Stomach
3.0
4
p.V560D
High
Small intestine 7.5
38
subunit) subunit) p.P577_D579del
subunit) Intermediate Stomach subunit)
5.2
1
Neither
870
Y. R. Kim et al. repeated the experiments twice, including tissue microdissection, PCR, SSCP, and sequencing analysis to ensure the specificity of the results, and found that the data were consistent (data not shown).
4. Discussion
Fig. 1 Mutations of CASP4 gene in GIST. SSCP (upper) and DNA sequencing analysis (lower) of CASP4 gene from tumors (lane T) and normal tissues (lane N) of the same patients. A, PCR product of CASP4 shows aberrant bands (arrows in lane T) as compared with SSCP from normal tissues (N). B, DNA sequencing from one of the aberrant band of the SSCP shows nucleotide substitution (C to G) in tumor tissue as compared with normal tissue.
PCR product was purified, and both strands were directly sequenced using a capillary automatic sequencer (ABI Prism Genetic Analyzer, Applied Biosystem).
3. Results Using the microdissection technique, we selectively procured tumor cells from histologic sections of 22 GISTs. Genomic DNA was isolated and analyzed for potential mutations in all coding exons of 11 CASP genes (CASP1-10 and 14) by PCR-SSCP analysis. All PCR products were clearly seen on the SSCP autoradiograms. Enrichment and direct sequence analysis of aberrantly migrating bands on the SSCP led to the identification of 4 mutations in 4 samples (18.2% [4/22]) (Table 1). None of the corresponding normal samples from the same patients showed evidence of mutations by SSCP (Fig. 1), indicating the mutations had a risen somatically (Fig. 1). There were 4 CASP4 mutations, which were all missense mutations, and were identical (c.1093CNG) in exon 8, which would result in amino acid substitution in the small protease subunit (p.L365V). We analyzed the relationship of the CASP mutations with the various clinicopathologic characteristics listed in Table 1. However, there was no significant association (Fisher exact test, P N .05). We
We have previously reported somatic mutations of CASP genes, including CASP1, 3, 4, 5, 6, 7, 8, 9, 10, and 14 in various human cancers [11-18]. Such a wide distribution of CASP gene mutations led us to further analyze mutations of CASP genes in GISTs, in which no data on CASP mutation has been reported. We analyzed the entire coding sequences of all human CASP genes and found that the CASP4 genes is somatically mutated in GISTs. This is the first report on CASP gene mutations in GIST, and the data show that GISTs occasionally harbor CASP gene mutations. One of the central aims of cancer research is to identify mutated genes that are causally implicated in carcinogenesis. Mutations in cancer could be categorized as either functional alterations affecting key genes underlying the neoplastic process or nonfunctional “passenger” changes [21]. In general, a high incidence, recurrent mutations, or functional derangements related to the characteristics of cancers may suggest that the mutated gene may be a cancer-related gene, but not a passenger gene [21]. We detected CASP4 mutations in 18.2% of the GISTs analyzed. Notably, all of the 4 CASP4 mutations detected in this study were identical and occurred in unrelated patients. This observation is in contrast to previous reports on apoptosis-related gene mutations, which showed that most of the mutations were missense mutations and were not identical [11-18]. One possible explanation could be that recurrent mutations were due to artificial errors, including contamination of the samples. This is unlikely because we analyzed KIT and PDGFRA in the same GIST tissues and found that the 4 GISTs tissues with the CASP4 mutation did not show the same mutational pattern of the KIT gene (Table 1). The Leu at amino acid residue 365 in caspase 4 is conserved in human caspase 3 but not in other caspases (Genbank database), suggesting a possibility that alteration of this residue is caspase 4 specific. However, whether the functional activity of the caspase 4 mutant is altered or not remains unknown. We previously analyzed CASP4 mutation in 337 common carcinomas, but there were only 2 mutations in the cancers (0.4%) [12]. Together, these data suggest that CASP4 gene mutation might be a feature of the GIST genome. The CASP4 mutations were not associated with clinicopathologic features of the GISTs (Table 1). One possible reason for the lack of association is that the CASP mutations are involved in an early stage of GIST development. In this study, we analyzed only 22 GISTs, which were the only GISTs available to us. Analysis of a small number of tissues might be misleading.
Caspase mutation in GIST Caspases are not only crucial in apoptosis but also in inflammation and differentiation [5-8]. In humans, inflammatory caspases are encoded by CASP1, CASP4, and CASP5. These caspases are called inflammatory caspases because the main caspase 1 substrates identified to date are pro–interleukin 1β and pro–interleukin 18, 2 related cytokines that play critical roles in inflammation [8]. Cancer cells produce many inflammatory mediators and interconnect with surrounding cells, and these play a role in the pathogenesis of cancers [8]. However, how the inflammatory caspases contribute to the pathogenesis of tumors remains unknown at this stage. There is no known study that connects GIST development and inflammation. Our study opens a possibility that CASP4 mutation might be involved in the pathogenesis of GIST. Although both KIT and PDGFRA mutations are key mechanisms in the development of GIST, other genetic alterations may cooperate with the KIT and PDGFRA mutations. Caspase 4 is involved in both apoptosis and inflammatory processes, and it could possibly contribute to GIST development. How caspase 4 mutation contributes to the pathogenesis of GIST needs to be addressed in future studies.
References [1] Reed JC. Mechanisms of apoptosis. Am J Pathol 2000;39:1415-30. [2] Nagata S. Apoptosis by death factor. Cell 1997;88:355-65. [3] Nicholson DW. Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ 1999;6:1028-42. [4] Kumar S. Mechanisms mediating caspase activation in cell death. Cell Death Differ 1999;6:1060-6. [5] Thornberry NA, Bull HG, Calaycay JR, et al. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature 1992;356:768-74.
871 [6] Munday NA, Vaillancourt JP, Ali A, et al. Molecular cloning and proapoptotic activity of ICErelII and ICErelIII, members of the ICE/CED3 family of cysteine proteases. J Biol Chem 1995;270:15870-6. [7] Kamens J, Paskind M, Huqunin M, et al. Identification and characterization of ICH-2, a novel member of the interleukin-1 betaconverting enzyme family of cysteine proteases. J Biol Chem 1995; 270:15250-6. [8] Martinon F, Tschopp J. Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases. Cell 2004;117: 561-74. [9] Hu S, Snipas SJ, Vincenz C, Salvesen G, Dixit VM. Caspase-14 is a novel developmentally regulated protease. J Biol Chem 1998;273: 29648-53. [10] Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100: 57-70. [11] Soung YH, Lee JW, Kim ST, et al. Somatic mutations of CASP3 gene in human cancers. Hum Genet 2004;115:112-5. [12] Soung YH, Jeong EG, Ahn CH, et al. Mutational analysis of caspase 1, 4, and 5 genes in common human cancers. HUM PATHOL 2008;39: 895-900. [13] Soung YH, Lee JW, Kim HS, et al. Inactivating mutations of CASPASE-7 gene in human cancers. Oncogene 2003;22:8048-52. [14] Kim HS, Lee JW, Soung YH, et al. Inactivating mutations of caspase-8 gene in colorectal carcinomas. Gastroenterology 2003;125:708-15. [15] Shin MS, Kim HS, Kang CS, et al. Inactivating mutations of CASP10 gene in non-Hodgkin lymphomas. Blood 2002;99:4094-9. [16] Lee JW, Kim MR, Soung YH, et al. Mutational analysis of the CASP6 gene in colorectal and gastric carcinomas. APMIS 2006;114:646-50. [17] Soung YH, Lee JW, Kim SY, et al. Mutational analysis of proapoptotic caspase-9 gene in common human carcinomas. APMIS 2006;114: 292-7. [18] Yoo NJ, Soung YH, Lee SH, Jeong EG, Lee SH. Mutational analysis of caspase-14 gene in common carcinomas. Pathology 2007;39:330-3. [19] Corless CL, Fletcher JA, Heinrich MC. Biology of gastrointestinal stromal tumors. J Clin Oncol 2004;22:3813-25. [20] Lee SH, Stehlik C, Reed JC. Cop, a caspase recruitment domaincontaining protein and inhibitor of caspase-1 activation processing. J Biol Chem 2001;276:34495-500. [21] Futreal PA, Coin L, Marshall M, et al. A census of human cancer genes. Nat Rev Cancer 2004;4:177-83.
www.elsevier.com/locate/humpath
Original contribution
Mutational analysis of CASP1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 14 genes in gastrointestinal stromal tumors☆ Yoo Ri Kim BS a , Kyoung Mee Kim MD b , Nam Jin Yoo MD a , Sug Hyung Lee MD a,⁎ a
Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Korea
b
Received 9 September 2008; revised 1 November 2008; accepted 7 November 2008
Keywords: Caspase; Mutation; GIST; Cancer; Apoptosis
Summary Deregulation of apoptosis is one of the hallmarks of cancer, and inactivation of cancer cell apoptosis has been reported in many cancers. Caspases, the main executioners during apoptosis and inflammation, have been reported to harbor inactivating mutations in several cancers. The aim of this study was to explore whether CASP1 to 10 and 14 genes that encode caspase 1 to 10 and 14 are somatically mutated in gastrointestinal stromal tumor. We analyzed the entire coding region and all splice sites of all 11 human CASP genes for the detection of somatic mutations in 22 gastrointestinal stromal tumors by a single strand conformation polymorphism assay. We found a recurrent CASP4 mutation (c.1093CNG [p.L365V]) in 4 gastrointestinal stromal tumors, but there were no mutations in the other 10 CASPs. The CASP4 mutation was a missense mutation and was predicted to substitute amino acids in the small protease subunit of caspase 4. Overall, the gastrointestinal stromal tumor tissues harbored a CASP mutation in 18.2% (4/22). Our data indicate that somatic mutation of the CASP4 gene is common in gastrointestinal stromal tumor and suggest a possibility that CASP4 mutation might lead to alteration of apoptotic or inflammatory function and contribute to the pathogenesis of some gastrointestinal stromal tumors. © 2009 Elsevier Inc. All rights reserved.
1. Introduction Apoptosis regulates normal tissue homeostasis, cellular differentiation, and development [1,2]. Apoptosis occurs through activation of a family of cysteine proteases that cleave the substrates at aspartate residues, known as caspases. The caspase family consists of 13 mammalian isoenzymes, of which 11 human isoenzymes are currently
☆ This work was supported by a grant from Korea Research Foundation in Korea (2007-314-E00042). ⁎ Corresponding author. E-mail address: [email protected] (S. H. Lee).
0046-8177/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2008.11.013
known [3,4]. The principal role of caspases is initiation and execution of apoptosis [3-4]. Caspases are classified as either effector or initiator caspases. The initiator caspases appear to be caspases 2, 8, 9, and 10, and the effector caspases appear to be caspases 3, 6, and 7 [3-4]. In addition, the interleukin 1β converting enzyme (caspase 1) subfamily caspases play a crucial role in both apoptosis and inflammation [3-8]. Caspase 14 is a unique caspase involved in cell differentiation and is not categorized into inflammatory or apoptotic caspases [9]. Inactivation of apoptosis allows cells to survive that are prone to genetic damage. Inactivation of apoptosis is one of the hallmarks of cancer [10]. Proapoptotic proteins are likely to be tumor suppressors, and inactivation of apoptosis by
Caspase mutation in GIST
869
somatic mutations has been reported in human cancers. In the caspase genes, somatic mutations of CASP3, CASP5, CASP7, CASP8, and CASP10 have frequently been detected in human cancer tissues [11-15], whereas those of CASP1, CASP4, CASP6, CASP9, and CASP14 have rarely been found [12,16-18]. Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors that are thought originate from the intestinal cells of Cajal [19]. GISTs are characterized by the expression of cKIT, a receptor tyrosine kinase. About 80% to 85% of GISTs harbor activating mutations of KIT or the platelet-derived growth factor receptor-α (PDGFRA) gene, which is considered to be the main mechanisms of GIST tumorigenesis [19]. Although most GISTs are responsive to imatinib (Gleevec) treatment, approximately 10% to 15% of GISTs show no response to imatinib (intrinsic or primary tumor cell resistance) [19]. Furthermore, GISTs do not respond to conventional chemotherapy considered standard for soft tissue sarcomas [19]. Together, these data suggest that GISTs might possess intrinsic mechanisms for apoptosis evasion. Also, alterations of inflammatory caspases are not known in GISTs. In this study, we analyzed 22 GISTs for the detection of somatic mutations in all types of caspase-encoding genes (CASP1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 14) that might contribute to the tumorigenesis of GISTs.
2. Materials and methods 2.1. Tissues and microdissection GIST tissues were obtained from 22 patients by surgical removal. Malignant and normal cells were selectively procured from hematoxylin and eosin-stained frozen sections using a 30G1/2 hypodermic needle (Becton Dickinson, Franklin Lakes, NJ) affixed to a micromanipulator by microdissection, as described previously [13-15]. DNA extraction was performed using a modified single-step DNA extraction method by proteinase K treatment, as described previously [13-15]. All of the patients were Asians
Table 1 Gene
(Korean). Approval was obtained from the institutional review board of College of Medicine, Catholic University of Korea, Seoul, Korea, for this study. The GISTs originated from stomach (n = 18), small intestine (n = 3), and esophagus (n = 1). Thirteen GISTs were less than 5 cm in diameter, and 9 were 5 to 10 cm in diameter. Fourteen GISTs showed less than 5 mitotic figures per 50 high-power fields, and 8 showed 5 to 10 mitotic figures per 50 high-power fields. The GISTs showed KIT mutations in 20 cases (20/22 [90.9%]). All of the KIT mutations were detected on exon 11, and they consisted of 11 deletions, 2 insertions, and 7 point mutations. Two GISTs showed neither KIT nor PDGFRA mutation. There were 4 high-risk, 7 intermediate-risk, and 11 low-risk GISTs.
2.2. Single strand conformation polymorphism analysis Genomic DNA from tumor cells and normal cells of corresponding patients was amplified with primer pairs covering the entire coding region of each CASP gene as described previously [11-18]. In the case of CASP1, we did not analyze exons 1 and 2 because the DNA sequence of CASP1 is exactly the same as that of the COP-1 gene [20]. Radioisotope was incorporated into the polymerase chain reaction (PCR) reactions for detection by autoradiogram. PCR and single strand conformation polymorphism (SSCP) analysis were performed as described previously [11-18]. After SSCP, DNA samples showing mobility shifts were cut from the dried gel and reamplified for 30 cycles using the same primer sets. DNA sequencing of the PCR products was carried out using a capillary automatic sequencer (ABI Prism Genetic Analyzer; Applied Biosystem, Foster City, CA) according to the manufacturer's recommendation.
2.3. Mutational analysis of KIT and PDGFRA Genomic DNA from the 22 GISTs was amplified by PCR with primer pairs covering exons 9, 11, 13, and 17 of the KIT gene and exons 12, 14, and 18 of the PDGFRA gene. Each
Summary of the CASP4 mutations in the GISTs Nucleotide change Exon (domain) (predicted amino acid change)
CASP4 c.1093CNG (p.L365V) CASP4 c.1093CNG (p.L365V) CASP4 c.1093CNG (p.L365V) CASP4 c.1093CNG (p.L365V) Abbreviation: HPF, high-power field.
8 (small protease 8 (small protease 8 (small protease 8 (small protease
Risk group
Tumor site
Size of Mitosis/50 KIT or PDGFRA tumor (cm) HPF mutation
Low
Stomach
5.0
0
p.Q575_R586dup
Low
Stomach
3.0
4
p.V560D
High
Small intestine 7.5
38
subunit) subunit) p.P577_D579del
subunit) Intermediate Stomach subunit)
5.2
1
Neither
870
Y. R. Kim et al. repeated the experiments twice, including tissue microdissection, PCR, SSCP, and sequencing analysis to ensure the specificity of the results, and found that the data were consistent (data not shown).
4. Discussion
Fig. 1 Mutations of CASP4 gene in GIST. SSCP (upper) and DNA sequencing analysis (lower) of CASP4 gene from tumors (lane T) and normal tissues (lane N) of the same patients. A, PCR product of CASP4 shows aberrant bands (arrows in lane T) as compared with SSCP from normal tissues (N). B, DNA sequencing from one of the aberrant band of the SSCP shows nucleotide substitution (C to G) in tumor tissue as compared with normal tissue.
PCR product was purified, and both strands were directly sequenced using a capillary automatic sequencer (ABI Prism Genetic Analyzer, Applied Biosystem).
3. Results Using the microdissection technique, we selectively procured tumor cells from histologic sections of 22 GISTs. Genomic DNA was isolated and analyzed for potential mutations in all coding exons of 11 CASP genes (CASP1-10 and 14) by PCR-SSCP analysis. All PCR products were clearly seen on the SSCP autoradiograms. Enrichment and direct sequence analysis of aberrantly migrating bands on the SSCP led to the identification of 4 mutations in 4 samples (18.2% [4/22]) (Table 1). None of the corresponding normal samples from the same patients showed evidence of mutations by SSCP (Fig. 1), indicating the mutations had a risen somatically (Fig. 1). There were 4 CASP4 mutations, which were all missense mutations, and were identical (c.1093CNG) in exon 8, which would result in amino acid substitution in the small protease subunit (p.L365V). We analyzed the relationship of the CASP mutations with the various clinicopathologic characteristics listed in Table 1. However, there was no significant association (Fisher exact test, P N .05). We
We have previously reported somatic mutations of CASP genes, including CASP1, 3, 4, 5, 6, 7, 8, 9, 10, and 14 in various human cancers [11-18]. Such a wide distribution of CASP gene mutations led us to further analyze mutations of CASP genes in GISTs, in which no data on CASP mutation has been reported. We analyzed the entire coding sequences of all human CASP genes and found that the CASP4 genes is somatically mutated in GISTs. This is the first report on CASP gene mutations in GIST, and the data show that GISTs occasionally harbor CASP gene mutations. One of the central aims of cancer research is to identify mutated genes that are causally implicated in carcinogenesis. Mutations in cancer could be categorized as either functional alterations affecting key genes underlying the neoplastic process or nonfunctional “passenger” changes [21]. In general, a high incidence, recurrent mutations, or functional derangements related to the characteristics of cancers may suggest that the mutated gene may be a cancer-related gene, but not a passenger gene [21]. We detected CASP4 mutations in 18.2% of the GISTs analyzed. Notably, all of the 4 CASP4 mutations detected in this study were identical and occurred in unrelated patients. This observation is in contrast to previous reports on apoptosis-related gene mutations, which showed that most of the mutations were missense mutations and were not identical [11-18]. One possible explanation could be that recurrent mutations were due to artificial errors, including contamination of the samples. This is unlikely because we analyzed KIT and PDGFRA in the same GIST tissues and found that the 4 GISTs tissues with the CASP4 mutation did not show the same mutational pattern of the KIT gene (Table 1). The Leu at amino acid residue 365 in caspase 4 is conserved in human caspase 3 but not in other caspases (Genbank database), suggesting a possibility that alteration of this residue is caspase 4 specific. However, whether the functional activity of the caspase 4 mutant is altered or not remains unknown. We previously analyzed CASP4 mutation in 337 common carcinomas, but there were only 2 mutations in the cancers (0.4%) [12]. Together, these data suggest that CASP4 gene mutation might be a feature of the GIST genome. The CASP4 mutations were not associated with clinicopathologic features of the GISTs (Table 1). One possible reason for the lack of association is that the CASP mutations are involved in an early stage of GIST development. In this study, we analyzed only 22 GISTs, which were the only GISTs available to us. Analysis of a small number of tissues might be misleading.
Caspase mutation in GIST Caspases are not only crucial in apoptosis but also in inflammation and differentiation [5-8]. In humans, inflammatory caspases are encoded by CASP1, CASP4, and CASP5. These caspases are called inflammatory caspases because the main caspase 1 substrates identified to date are pro–interleukin 1β and pro–interleukin 18, 2 related cytokines that play critical roles in inflammation [8]. Cancer cells produce many inflammatory mediators and interconnect with surrounding cells, and these play a role in the pathogenesis of cancers [8]. However, how the inflammatory caspases contribute to the pathogenesis of tumors remains unknown at this stage. There is no known study that connects GIST development and inflammation. Our study opens a possibility that CASP4 mutation might be involved in the pathogenesis of GIST. Although both KIT and PDGFRA mutations are key mechanisms in the development of GIST, other genetic alterations may cooperate with the KIT and PDGFRA mutations. Caspase 4 is involved in both apoptosis and inflammatory processes, and it could possibly contribute to GIST development. How caspase 4 mutation contributes to the pathogenesis of GIST needs to be addressed in future studies.
References [1] Reed JC. Mechanisms of apoptosis. Am J Pathol 2000;39:1415-30. [2] Nagata S. Apoptosis by death factor. Cell 1997;88:355-65. [3] Nicholson DW. Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ 1999;6:1028-42. [4] Kumar S. Mechanisms mediating caspase activation in cell death. Cell Death Differ 1999;6:1060-6. [5] Thornberry NA, Bull HG, Calaycay JR, et al. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature 1992;356:768-74.
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