© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12193
APMIS 122: 499–504
Mutational and expressional analysis of SMC2 gene in gastric and colorectal cancers with microsatellite instability EUN MI JE,1 NAM JIN YOO1,2 and SUG HYUNG LEE1,2 1
Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, 2Cancer Evolution Research Center, The Catholic University of Korea, Seoul, Korea
Je EM, Yoo NJ, Lee SH. Mutational and expressional analysis of SMC2 gene in gastric and colorectal cancers with microsatellite instability. APMIS 2014; 122: 499–504. Structural maintenance of chromosomes 2 (SMC2) gene encodes condensin complexes that are required for proper chromosome segregation and maintenance of chromosomal stability. Although cells with defective chromosome segregation become aneuploid and are prone to harbor chromosome instability, pathologic implications of SMC2 gene alterations are largely unknown. In a public database, we found that SMC2 gene had mononucleotide repeats that could be mutated in cancers with microsatellite instability (MSI). In this study, we analyzed these repeats in 32 gastric cancers (GC) with high MSI (MSI-H), 59 GC with low MSI (MSI-L)/stable MSI (MSS), 43 colorectal cancers (CRC) with MSI-H and 60 CRC with MSI-L/MSS by single-strand conformation polymorphism (SSCP) and DNA sequencing. We also analyzed SMC2 protein expression in GC and CRC tissues using immunohistochemistry. We found SMC2 frameshift mutations in two GC and two CRC that would result in truncation of SMC2. The mutations were detected exclusively in MSI-H cancers, but not in MSI-L/MSS cancers. Loss of SMC2 expression was observed in 22% of GC and 25% of CRC. Of note, all of the cancers with SMC2 frameshift mutations displayed loss of SMC2 expression. Also, both GC and CRC with MSI-H had significantly higher incidences in SMC2 frameshift mutations and loss of SMC2 expression than those with MSI-L/MSS. Our data indicate that SMC2 gene is altered by both frameshift mutation and loss of expression in GC and CRC with MSI-H, and suggest that SMC2 gene alterations might be involved in pathogenesis of these cancers. Key words: SMC2; condensin; frameshift mutation; SMC2 expression; cancer; microsatellite instability. Sug Hyung Lee, Department of Pathology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea. e-mail: [email protected]
In mitosis, sister chromatids are physically linked together before the segregation at anaphase. Condensins are large protein complexes that play a central role in chromosome assembly and condensation, and essential for proper segregation of sister chromatids (1–4). Eukaryotic cells have two types of condensin complexes, known as condensin I and II. Both complexes share structural maintenance of chromosomes 2 (SMC2) and SMC4 as core subunits, both belonging to a large family of DNA-binding ATPases (2). It is well known that cells that are defective for proper chromosome segregation become aneuploid, which induces chromosome
Received 31 May 2013. Accepted 5 August 2013
instability and is a characteristic of many types of tumorigenesis (4). Experimentally, depletion of SMC2 resulted in changes in chromosome morphology and inaccurate segregation during mitosis (5). In human tissues, somatic mutations of SMC2 gene have been reported in pyothorax-associated lymphomas (5). Cells harboring SMC2 mutations showed features with chromosomal destabilization as well, suggesting that impairment of condensin functions by somatic mutation of SMC2 might induce genome instability and contribute to tumorigenesis. In a public genome database (http://genome.cse. ucsc.edu/), we found that human SMC2 gene has mononucleotide repeats in the coding sequences that could be targets for frameshift mutation in 499
JE et al.
cancers with microsatellite instability (MSI). Frameshift mutations of genes containing mononucleotide repeats are features of gastric (GC) and colorectal cancers (CRC) with MSI (6–10). It is possible that frameshift mutations of the SMC2-encoding genes could cause alterations in SMC2 functions and contribute to cancer pathogenesis in the cancers with MSI. However, the data on mutations in SMC2 gene are known only in the pyothorax-associated lymphomas (5). In this study, we used tumors that had already been evaluated their MSI status (11). With these tumor tissues, we explored whether the SMC2 gene is somatically mutated in GC and CRC with MSI. MATERIALS AND METHODS Tissue samples For the mutation analysis, methacarn-fixed tissues of sporadic 91 GC and 103 CRC were used in this study (Table 1). All of the cancer patients were Koreans. The GC consisted of 32 with high MSI (MSI-H), 14 with low MSI (MSI-L) and 45 with stable MSI (MSS). The CRC consisted of 43 with MSI-H, 15 with MSI-L and 45 with MSS. MSI status of these tumors had been evaluated using a panel of five mononucleotide repeat sequences (BAT25, BAT26, NR-21, NR-24 and MONO-27) and three dinucleotide repeat sequences (D2S123, D5S346 and D17S250). The tumors may be characterized as: MSI-H, if three or more of these markers show
instability, and MSI-L, if one or two of the markers shows instability (11). Approval for this study was obtained from the Institutional Review Board of Catholic University of Korea. Tumor cells and corresponding normal cells from the same patients were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle affixed to a micromanipulator, as described previously (12–14). DNA extraction was performed by a modified single-step DNA extraction method (12–14).
Single-strand conformation polymorphism (SSCP) analysis for mutation detection SMC2 exon 6 (two A7 repeats), exon 9 (A7) and exon 17 (A7) have mononucleotide repeats in their coding sequences. Genomic DNA from the microdissected cells was isolated, and was amplified by polymerase chain reaction (PCR) with three specific primer pairs (Table 2). Radioisotope ([32P]dCTP) was incorporated into PCR products for detection by autoradiography. Procedures of PCR and SSCP analysis were performed as described previously (12–14). After SSCP, mobility shifts on the SSCP gels (FMC Mutation Detection Enhancement system; Intermountain Scientific, Kaysville, UT, USA) were determined by visual inspection. Direct DNA sequencing reactions were performed in the cancers with the mobility shifts in the SSCP. Sequencing of the PCR products was carried out using a capillary automatic sequencer (3730 DNA Analyzer, Applied Biosystem, Carlsbad, CA, USA). When mutations in the SMC2 genes were suspected by SSCP, analysis of an independently isolated DNA from another tissue section of the same patients was performed to exclude potential artifacts originated from PCR.
Table 1. Summary of pathologic features of gastric and colorectal cancers No. of gastric carcinomas No. of colorectal carcinomas MSI-H (n = 43) MSI-H (n = 32) MSS/MSI-L (n = 59) TNM TNM I 13 19 I 7 II 11 24 II 15 III 7 13 III 18 IV 1 3 IV 3 Lauren’s subtype Location (colon) Diffuse 20 33 Cecum 6 Intestinal 12 26 Ascending 24 Transverse 10 EGC vs AGC EGC 3 5 Descending & sigmoid 3 AGC 29 54 Rectum 0 EGC, early gastric cancer; AGC, advanced gastric cancer; TNM, tumor, lymph node, metastasis.
MSS/MSI-L (n = 60) 9 21 25 5 0 4 4 21 31
Table 2. Primer sequences of the SMC2 gene used in this study Gene Sequences
Size (bp)
SMC2 Exon 6
129
Annealing temperature (°C) 47
120
49
152
49
SMC2 Exon 9 SMC2 Exon 17
500
F: 5′-TCATTCCAGATTTTATCCA-3′ R: 5′-ACCGTCTTAATTTCTTTCAG-3′ F: 5′-GCTCAGCGAGTTAATACTAAATC-3′ R: 5′-ATTAAGCTCACTTACCTCAACC-3′ F: 5′-TGGGAGATGAAAACTGAA-3′ R: 5′-ACGGATATGAATAAAACCAG-3′
© 2014 APMIS. Published by John Wiley & Sons Ltd
SMC2 MUTATIONS IN GASTRIC AND COLORECTAL CANCERS
Immunohistochemistry Using the sections from GC and CRC tissues, immunohistochemistry for SMC2, which showed most frequent mutations, was performed in this study. The tissues consisted of 32 GC and 43 CRC with MSI-H, and 59 GC and 60 CRC with MSI-L/MSS. We used ImmPRESS System (Vector Laboratories, Burlinggame, CA, USA) with rabbit polyclonal antibody against human SMC2 (ab10412) (Abcam, Cambridge, UK; dilution 1/200). After deparaffinization, 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 immunohistochemical procedure was performed as described previously (15). The reaction products were developed with diaminobenzidine and counterstained with hematoxylin. By visual inspection under light microscope, tumors were interpreted as positive when 20–100% of the cells showed moderate to intense staining in cytoplasm or nucleus, and as negative when 0–19% of the cells showed no or weak or moderate or intense staining by immunohistochemistry. Intensity of SMC2 immunostaining was interpreted as positive or negative independently by two pathologists. On the initial observation, two pathologists had agreement on the interpretation (Cohen’s kappa coefficient; 0.86). For the remaining cases, they finally reached to complete agreement. The immunostaining was judged to be specific by absence of consistent immunostaining of cells with replacement of primary antibody with the blocking reagent and reduction of immunostaining intensity as dilution of the antibody was increased. For the statistical analysis of the immunohistochemical data, we used Fisher’s exact test and chi-squared test.
gous according to the SSCP and direct sequencing analyses (Fig. 1). These four mutations were deletion mutations of one base in the repeats that would cause premature stop codons, which lead to the termination of translation (Table 3). There was no aberrant band in the SSCP of exon 9. All of the mutations were detected in the cancers with MSI-H (Table 3), but not in those with MSIL or MSS. There were statistical differences between the frequency of the mutation in the cancers with MSI-H (4/75) and MSI-L/MSS (0/119) (Fisher’s exact test, p = 0.021). Two GC (2/32; 6.3%) and two CRC (2/43; 4.7%) with MSI-H
A
B
RESULTS SMC2 gene mutations
Of the 91 GC and 103 CRC, we found aberrant SSCP bands in two GC and two CRC (Fig. 1; Table 3). Direct DNA sequencing analysis of cancer tissues with the aberrantly migrating bands led to identification of SMC2 gene mutations in A7 of exon 6 (one GC) and A7 of exon 17 (one GC and two CRC) (Table 3). DNA from normal tissues from the same patients showed no evidence of the mutation in both SSCP and DNA sequencing, indicating the mutations had risen somatically (Fig. 1). All of the mutations were interpreted as heterozy-
Fig. 1. Representative single-strand conformation polymorphism (SSCP) and DNA sequencing of SMC2 gene in the cancers. (A) In the SSCP, the arrows (Lane T) indicate aberrant bands compared to the SSCP from normal tissues (N). (B) Direct DNA sequencing analyses of exon 6 (left) and exon 17 (right) show heterozygous deletion of a nucleotide in tumor tissue as compared to normal tissues.
Table 3. Summary of the SMC2 frameshift mutations in gastric and colorectal cancers with MSI Incidence in cancers Gene Location Repeats Repeats MSI status with MSI-H (%) (wild type) (Mutation) of the mutation cases (n) SMC2 Exon 6 A7 A6 MSI-H (1) Gastric: 1/32 (3.1) Colorectal: 0/43 (0) SMC2 Exon 17 A7 A6 MSI-H (3) Gastric: 1/32 (3.1) Colorectal: 2/43 (4.7) © 2014 APMIS. Published by John Wiley & Sons Ltd
Nucleotide change (predicted amino acid change) c.563delA (p.Lys188Argfsx5) c. 2251delA (p.Thr751Profsx9)
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A
B
C
D
Fig. 2. Visualization of structural maintenance of chromosomes 2 (SMC2) expression in gastric and colorectal cancer tissues with MSI-H by immunohistochemistry. (A) A gastric cancer without mutation in the evaluated SMC2 repeats shows SMC2 immunostaining in the nuclei of cancer cells. (B) Another gastric cancer with a frameshift mutation in a repeat shows negative SMC2 immunostaining in the cancer cells. (C) A colon cancer without mutation in the evaluated SMC2 repeats shows SMC2 immunostaining in the cancer cells. (D) Another colon cancer with a frameshift mutation in a repeat shows negative SMC2 immunostaining in the cancer cells (scale bars: 20 lm).
harbored the mutations. There was no significant association of the mutations with the clinicopathologic data of the patient (age, sex, stage and metastasis). There was no correlation between histologic features of the tumors (histologic grade, subtypes, mucinous histology, medullary pattern and tumorinfiltrating lymphocytes) and the presence of the mutations, either. Expression of SMC2 protein
We analyzed SMC2 protein expression in 32 GC and 43 CRC with MSI-H, and 59 GC and 60 CRC with MSI-L/MSS by immunohistochemistry. Immunopositivity for SMC2 was observed in 71 (78.0%) of the total 91 GC and 77 (74.8%) of the total 103 CRC. In the cancers with MSI-H, the immunopositivity was observed in 65.6% of the GC and 62.8% of the CRC (Fig. 2; Table 4). Of the four cancers with SMC2 frameshift mutations, none of them showed positive SMC2 immunostaining, and there was a significant difference of SMC2 expression between MSI-H cancers harboring mutations in SMC2 mutations and those with MSI-H that have not acquired mutations in SMC2 (Fisher’s exact test, p = 0.014). There were significant differences in SMC2 immunopositivity between the GC with MSI-H and the GC with MSI-L/MSS (Fisher’s exact test, p = 0.025), and between the CRC with MSI-H and the CRC with MSI-L/MSS (Fisher’s exact test, p = 0.012; Table 4). The immunostaining 502
Table 4. Summary of the SMC2 expression in gastric and colorectal cancers Positive SMC2 expression (%) GC with MSI-H (n = 32) 21 (65.6) CRC with MSI-H (n = 43) 27 (62.8) GC with MSI-L/MSS (n = 59) 50 (84.7) CRC with MSI-L/MSS (n = 60) 50 (83.3) MSI-H GC and CRC with 0 (0) SMC2 mutation (n = 4) MSI-H GC and CRC without 48 (67.6) SMC2 mutation (n = 71)
of SMC2, when present, was observed mainly in nuclei and less weakly in cytoplasm.
DISCUSSION Despite the importance of condensin functions in maintaining genome instability (2), genetic alterations of genes encoding components in condensin complexes are rarely known in human cancers. Based on the earlier report that showed SMC2 gene mutations in lymphomas with chromosomal destabilization (5), we attempted to address whether somatic frameshift mutation of SMC2 was present in GC and CRC tissues. We found that the mononucleotide repeats in coding region of SMC2 harbored frameshift mutations in both GC and CRC. The mutations were detected © 2014 APMIS. Published by John Wiley & Sons Ltd
SMC2 MUTATIONS IN GASTRIC AND COLORECTAL CANCERS
exclusively in the cancers with MSI-H, but not in those with MSI-L or MSS, indicating SMC2 gene is altered in GC and CRC with MSI-H by somatic frameshift mutations. In the present study, we detected two types of SMC2 mutations, both of which were novel mutations. Both mutations would cause change or delete amino acids after the frameshift mutation sites and hence resemble a typical loss-of-function mutation. The mutations at exon 6 and exon 17 would remove amino acids after the 188th residue and the 751th residues, respectively (Table 3). SMC2 protein is 1 198 amino acid-long and consists of N-terminal ATPase domain, N-terminal coiled-coil domain, hinge domain, C-terminal coiled-coil domain and C-terminal ATPase domain. The mutation at exon 6 would remove N-terminal coiled-coil domain, hinge domain, C-terminal coiled-coil domain and C-terminal ATPase domain, while the mutation at exon 17 would remove C-terminal coiled-coil domain and C-terminal ATPase domain. In cohesion I and II, SMC2 binds other SMC proteins and forms heterodimer for their functions (1, 2). It is likely that the deleted mutants would impair heterodimer formation and hamper its functions in maintaining chromosomal stability. We observed that SMC2 immunostaining was very weakly detectable or not detectable in all of the cancers with SMC2 frameshift mutations (Table 4). Because the antibody against SMC2 had been raised by a peptide immunogen within amino acid residues encoded by exon 27 that would be removed by the SMC2 frameshift mutations detected in the present study, the mutated SMC2 proteins might not be detected in cancer cells by this antibody. SSCP and direct sequencing of SMC2 in the mutated cases displayed that both of the frameshift mutations were heterozygous ones, suggesting that one allele was intact (Fig. 1). Loss of the SMC2 immunostaining in the cancers might be caused by the mutation in one allele and by the other gene silencing mechanisms in the second allele. Another possibility could be that quantity of SMC2 expression from one allele might not be enough to be detected in the immunohistochemistry. Of note, about one-third (23/71) of tumors even without SMC2 mutation showed loss of SMC2 expression by the immunohistochemistry (Table 4). These data suggest a possibility that loss of SMC2 expression in GC and CRC may be affected not only by frameshift mutations in the repeats, but also by other mechanisms such as epigenetic mechanisms or other somatic mutations outside of the frameshift mutation sites. Although condensin is important in maintaining chromosome stability, its implication in tumori-
© 2014 APMIS. Published by John Wiley & Sons Ltd
genesis has largely been unknown. An earlier study described that SMC2 was overexpressed in about 70% of CRC and the remaining 30% CRC were devoid of SMC2 expression (16). This report was in agreement with our data that showed loss of SMC2 expression in both CRC and GC. Functionally, depletion of SMC2 altered chromosome structure (2, 5). However, overexpression of C-terminus of SMC2 also disturbed chromosome condensation and mitotic progression (17). It should be studied in future whether overexpression or loss of expression or both of SMC2 is widespread in human cancers and whether consequences of each alteration are related to clinical phenotypes of cancers. Also, it remains to be clarified how the functions of SMC2 are different between cancers with MSI and without MSI, and what is the consequence of its frameshift mutations in their pathogenesis. In summary, our data indicate that SMC2 gene is altered in GC and CRC with MSI by frameshift mutation and loss of expression, and suggest that these alterations might contribute to pathogenesis of the cancers with MSI.
This work was supported by funding from Korea Research Foundation (2012R1A5A2047939).
REFERENCES 1. Cuylen S, Metz J, Haering CH. Condensin structures chromosomal DNA through topological links. Nat Struct Mol Biol 2011;18:894–901. 2. Nasmyth K, Haering CH. The structure and function of SMC and kleisin complexes. Annu Rev Biochem 2005;74:595–648. 3. Akai Y, Kurokawa Y, Nakazawa N, Tonami-Murakami Y, Suzuki Y, Yoshimura SH, et al. Opposing role of condensin hinge against replication protein A in mitosis and interphase through promoting DNA annealing. Open Biol 2011;1:110023. 4. Sen S. Aneuploidy and cancer. Curr Opin Oncol 2000;12:82–8. 5. Ham MF, Takakuwa T, Rahadiani N, Tresnasari K, Nakajima H, Aozasa K. Condensin mutations and abnormal chromosomal structures in pyothorax-associated lymphoma. Cancer Sci 2007;98:1041–7. 6. Imai K, Yamamoto H. Carcinogenesis and microsatellite instability: the interrelationship between genetics and epigenetics. Carcinogenesis 2008;29:673–80. 7. Grady WM. Genomic instability and colon cancer. Cancer Metastasis Rev 2004;23:11–27. 8. Gorringe KL, Chin SF, Pharoah P, Staines JM, Oliveira C, Edwards PA, et al. Evidence that both genetic instability and selection contribute to the accumulation of chromosome alterations in cancer. Carcinogenesis 2005;26:923–30.
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9. Markowitz S, Wang J, Myeroff L, Parsons R, Sun L, Lutterbaugh J, et al. Inactivation of the type II TGFbeta receptor in colon cancer cells with microsatellite instability. Science 1995;268:1336–8. 10. Rampino N, Yamamoto H, Ionov Y, Li Y, Sawai H, Reed JC, et al. Somatic frameshift mutations in the BAX gene in colon cancers of the microsatellite mutator phenotype. Science 1997;275:967–9. 11. Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature 2012;487:330–7. 12. Kim YR, Oh JE, Kim MS, Kang MR, Park SW, Han JY, et al. Oncogenic NRF2 mutations in squamous cell carcinomas of oesophagus and skin. J Pathol 2010;220:446–51. 13. Lee JW, Soung YH, Kim SY, Lee HW, Park WS, Nam SW, et al. PIK3CA gene is frequently mutated in breast carcinomas and hepatocellular carcinomas. Oncogene 2005;24:1477–80.
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14. Kim MS, Hur SY, Yoo NJ, Lee SH. Mutational analysis of FOXL2 codon 134 in granulosa cell tumour of ovary and other human cancers. J Pathol 2010;221:147–52. 15. Kim MS, Oh JE, Kim YR, Park SW, Kang MR, Kim SS, et al. Somatic mutations and losses of expression of microRNA regulation-related genes AGO2 and TNRC6A in gastric and colorectal cancers. J Pathol 2010;221:139–46. 16. D avalos V, S uarez-L opez L, Casta~ no J, Messent A, Abasolo I, Fernandez Y, et al. Human SMC2 protein, a core subunit of human condensin complex, is a novel transcriptional target of the WNT signaling pathway and a new therapeutic target. J Biol Chem 2012;287:43472–81. 17. Zhai L, Wang H, Tang W, Liu W, Hao S, Zeng X. Disturbance in function and expression of condensin affects chromosome compaction in HeLa cells. Cell Biol Int 2011;35:735–40.
© 2014 APMIS. Published by John Wiley & Sons Ltd
APMIS 122: 499–504
Mutational and expressional analysis of SMC2 gene in gastric and colorectal cancers with microsatellite instability EUN MI JE,1 NAM JIN YOO1,2 and SUG HYUNG LEE1,2 1
Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, 2Cancer Evolution Research Center, The Catholic University of Korea, Seoul, Korea
Je EM, Yoo NJ, Lee SH. Mutational and expressional analysis of SMC2 gene in gastric and colorectal cancers with microsatellite instability. APMIS 2014; 122: 499–504. Structural maintenance of chromosomes 2 (SMC2) gene encodes condensin complexes that are required for proper chromosome segregation and maintenance of chromosomal stability. Although cells with defective chromosome segregation become aneuploid and are prone to harbor chromosome instability, pathologic implications of SMC2 gene alterations are largely unknown. In a public database, we found that SMC2 gene had mononucleotide repeats that could be mutated in cancers with microsatellite instability (MSI). In this study, we analyzed these repeats in 32 gastric cancers (GC) with high MSI (MSI-H), 59 GC with low MSI (MSI-L)/stable MSI (MSS), 43 colorectal cancers (CRC) with MSI-H and 60 CRC with MSI-L/MSS by single-strand conformation polymorphism (SSCP) and DNA sequencing. We also analyzed SMC2 protein expression in GC and CRC tissues using immunohistochemistry. We found SMC2 frameshift mutations in two GC and two CRC that would result in truncation of SMC2. The mutations were detected exclusively in MSI-H cancers, but not in MSI-L/MSS cancers. Loss of SMC2 expression was observed in 22% of GC and 25% of CRC. Of note, all of the cancers with SMC2 frameshift mutations displayed loss of SMC2 expression. Also, both GC and CRC with MSI-H had significantly higher incidences in SMC2 frameshift mutations and loss of SMC2 expression than those with MSI-L/MSS. Our data indicate that SMC2 gene is altered by both frameshift mutation and loss of expression in GC and CRC with MSI-H, and suggest that SMC2 gene alterations might be involved in pathogenesis of these cancers. Key words: SMC2; condensin; frameshift mutation; SMC2 expression; cancer; microsatellite instability. Sug Hyung Lee, Department of Pathology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea. e-mail: [email protected]
In mitosis, sister chromatids are physically linked together before the segregation at anaphase. Condensins are large protein complexes that play a central role in chromosome assembly and condensation, and essential for proper segregation of sister chromatids (1–4). Eukaryotic cells have two types of condensin complexes, known as condensin I and II. Both complexes share structural maintenance of chromosomes 2 (SMC2) and SMC4 as core subunits, both belonging to a large family of DNA-binding ATPases (2). It is well known that cells that are defective for proper chromosome segregation become aneuploid, which induces chromosome
Received 31 May 2013. Accepted 5 August 2013
instability and is a characteristic of many types of tumorigenesis (4). Experimentally, depletion of SMC2 resulted in changes in chromosome morphology and inaccurate segregation during mitosis (5). In human tissues, somatic mutations of SMC2 gene have been reported in pyothorax-associated lymphomas (5). Cells harboring SMC2 mutations showed features with chromosomal destabilization as well, suggesting that impairment of condensin functions by somatic mutation of SMC2 might induce genome instability and contribute to tumorigenesis. In a public genome database (http://genome.cse. ucsc.edu/), we found that human SMC2 gene has mononucleotide repeats in the coding sequences that could be targets for frameshift mutation in 499
JE et al.
cancers with microsatellite instability (MSI). Frameshift mutations of genes containing mononucleotide repeats are features of gastric (GC) and colorectal cancers (CRC) with MSI (6–10). It is possible that frameshift mutations of the SMC2-encoding genes could cause alterations in SMC2 functions and contribute to cancer pathogenesis in the cancers with MSI. However, the data on mutations in SMC2 gene are known only in the pyothorax-associated lymphomas (5). In this study, we used tumors that had already been evaluated their MSI status (11). With these tumor tissues, we explored whether the SMC2 gene is somatically mutated in GC and CRC with MSI. MATERIALS AND METHODS Tissue samples For the mutation analysis, methacarn-fixed tissues of sporadic 91 GC and 103 CRC were used in this study (Table 1). All of the cancer patients were Koreans. The GC consisted of 32 with high MSI (MSI-H), 14 with low MSI (MSI-L) and 45 with stable MSI (MSS). The CRC consisted of 43 with MSI-H, 15 with MSI-L and 45 with MSS. MSI status of these tumors had been evaluated using a panel of five mononucleotide repeat sequences (BAT25, BAT26, NR-21, NR-24 and MONO-27) and three dinucleotide repeat sequences (D2S123, D5S346 and D17S250). The tumors may be characterized as: MSI-H, if three or more of these markers show
instability, and MSI-L, if one or two of the markers shows instability (11). Approval for this study was obtained from the Institutional Review Board of Catholic University of Korea. Tumor cells and corresponding normal cells from the same patients were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle affixed to a micromanipulator, as described previously (12–14). DNA extraction was performed by a modified single-step DNA extraction method (12–14).
Single-strand conformation polymorphism (SSCP) analysis for mutation detection SMC2 exon 6 (two A7 repeats), exon 9 (A7) and exon 17 (A7) have mononucleotide repeats in their coding sequences. Genomic DNA from the microdissected cells was isolated, and was amplified by polymerase chain reaction (PCR) with three specific primer pairs (Table 2). Radioisotope ([32P]dCTP) was incorporated into PCR products for detection by autoradiography. Procedures of PCR and SSCP analysis were performed as described previously (12–14). After SSCP, mobility shifts on the SSCP gels (FMC Mutation Detection Enhancement system; Intermountain Scientific, Kaysville, UT, USA) were determined by visual inspection. Direct DNA sequencing reactions were performed in the cancers with the mobility shifts in the SSCP. Sequencing of the PCR products was carried out using a capillary automatic sequencer (3730 DNA Analyzer, Applied Biosystem, Carlsbad, CA, USA). When mutations in the SMC2 genes were suspected by SSCP, analysis of an independently isolated DNA from another tissue section of the same patients was performed to exclude potential artifacts originated from PCR.
Table 1. Summary of pathologic features of gastric and colorectal cancers No. of gastric carcinomas No. of colorectal carcinomas MSI-H (n = 43) MSI-H (n = 32) MSS/MSI-L (n = 59) TNM TNM I 13 19 I 7 II 11 24 II 15 III 7 13 III 18 IV 1 3 IV 3 Lauren’s subtype Location (colon) Diffuse 20 33 Cecum 6 Intestinal 12 26 Ascending 24 Transverse 10 EGC vs AGC EGC 3 5 Descending & sigmoid 3 AGC 29 54 Rectum 0 EGC, early gastric cancer; AGC, advanced gastric cancer; TNM, tumor, lymph node, metastasis.
MSS/MSI-L (n = 60) 9 21 25 5 0 4 4 21 31
Table 2. Primer sequences of the SMC2 gene used in this study Gene Sequences
Size (bp)
SMC2 Exon 6
129
Annealing temperature (°C) 47
120
49
152
49
SMC2 Exon 9 SMC2 Exon 17
500
F: 5′-TCATTCCAGATTTTATCCA-3′ R: 5′-ACCGTCTTAATTTCTTTCAG-3′ F: 5′-GCTCAGCGAGTTAATACTAAATC-3′ R: 5′-ATTAAGCTCACTTACCTCAACC-3′ F: 5′-TGGGAGATGAAAACTGAA-3′ R: 5′-ACGGATATGAATAAAACCAG-3′
© 2014 APMIS. Published by John Wiley & Sons Ltd
SMC2 MUTATIONS IN GASTRIC AND COLORECTAL CANCERS
Immunohistochemistry Using the sections from GC and CRC tissues, immunohistochemistry for SMC2, which showed most frequent mutations, was performed in this study. The tissues consisted of 32 GC and 43 CRC with MSI-H, and 59 GC and 60 CRC with MSI-L/MSS. We used ImmPRESS System (Vector Laboratories, Burlinggame, CA, USA) with rabbit polyclonal antibody against human SMC2 (ab10412) (Abcam, Cambridge, UK; dilution 1/200). After deparaffinization, 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 immunohistochemical procedure was performed as described previously (15). The reaction products were developed with diaminobenzidine and counterstained with hematoxylin. By visual inspection under light microscope, tumors were interpreted as positive when 20–100% of the cells showed moderate to intense staining in cytoplasm or nucleus, and as negative when 0–19% of the cells showed no or weak or moderate or intense staining by immunohistochemistry. Intensity of SMC2 immunostaining was interpreted as positive or negative independently by two pathologists. On the initial observation, two pathologists had agreement on the interpretation (Cohen’s kappa coefficient; 0.86). For the remaining cases, they finally reached to complete agreement. The immunostaining was judged to be specific by absence of consistent immunostaining of cells with replacement of primary antibody with the blocking reagent and reduction of immunostaining intensity as dilution of the antibody was increased. For the statistical analysis of the immunohistochemical data, we used Fisher’s exact test and chi-squared test.
gous according to the SSCP and direct sequencing analyses (Fig. 1). These four mutations were deletion mutations of one base in the repeats that would cause premature stop codons, which lead to the termination of translation (Table 3). There was no aberrant band in the SSCP of exon 9. All of the mutations were detected in the cancers with MSI-H (Table 3), but not in those with MSIL or MSS. There were statistical differences between the frequency of the mutation in the cancers with MSI-H (4/75) and MSI-L/MSS (0/119) (Fisher’s exact test, p = 0.021). Two GC (2/32; 6.3%) and two CRC (2/43; 4.7%) with MSI-H
A
B
RESULTS SMC2 gene mutations
Of the 91 GC and 103 CRC, we found aberrant SSCP bands in two GC and two CRC (Fig. 1; Table 3). Direct DNA sequencing analysis of cancer tissues with the aberrantly migrating bands led to identification of SMC2 gene mutations in A7 of exon 6 (one GC) and A7 of exon 17 (one GC and two CRC) (Table 3). DNA from normal tissues from the same patients showed no evidence of the mutation in both SSCP and DNA sequencing, indicating the mutations had risen somatically (Fig. 1). All of the mutations were interpreted as heterozy-
Fig. 1. Representative single-strand conformation polymorphism (SSCP) and DNA sequencing of SMC2 gene in the cancers. (A) In the SSCP, the arrows (Lane T) indicate aberrant bands compared to the SSCP from normal tissues (N). (B) Direct DNA sequencing analyses of exon 6 (left) and exon 17 (right) show heterozygous deletion of a nucleotide in tumor tissue as compared to normal tissues.
Table 3. Summary of the SMC2 frameshift mutations in gastric and colorectal cancers with MSI Incidence in cancers Gene Location Repeats Repeats MSI status with MSI-H (%) (wild type) (Mutation) of the mutation cases (n) SMC2 Exon 6 A7 A6 MSI-H (1) Gastric: 1/32 (3.1) Colorectal: 0/43 (0) SMC2 Exon 17 A7 A6 MSI-H (3) Gastric: 1/32 (3.1) Colorectal: 2/43 (4.7) © 2014 APMIS. Published by John Wiley & Sons Ltd
Nucleotide change (predicted amino acid change) c.563delA (p.Lys188Argfsx5) c. 2251delA (p.Thr751Profsx9)
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B
C
D
Fig. 2. Visualization of structural maintenance of chromosomes 2 (SMC2) expression in gastric and colorectal cancer tissues with MSI-H by immunohistochemistry. (A) A gastric cancer without mutation in the evaluated SMC2 repeats shows SMC2 immunostaining in the nuclei of cancer cells. (B) Another gastric cancer with a frameshift mutation in a repeat shows negative SMC2 immunostaining in the cancer cells. (C) A colon cancer without mutation in the evaluated SMC2 repeats shows SMC2 immunostaining in the cancer cells. (D) Another colon cancer with a frameshift mutation in a repeat shows negative SMC2 immunostaining in the cancer cells (scale bars: 20 lm).
harbored the mutations. There was no significant association of the mutations with the clinicopathologic data of the patient (age, sex, stage and metastasis). There was no correlation between histologic features of the tumors (histologic grade, subtypes, mucinous histology, medullary pattern and tumorinfiltrating lymphocytes) and the presence of the mutations, either. Expression of SMC2 protein
We analyzed SMC2 protein expression in 32 GC and 43 CRC with MSI-H, and 59 GC and 60 CRC with MSI-L/MSS by immunohistochemistry. Immunopositivity for SMC2 was observed in 71 (78.0%) of the total 91 GC and 77 (74.8%) of the total 103 CRC. In the cancers with MSI-H, the immunopositivity was observed in 65.6% of the GC and 62.8% of the CRC (Fig. 2; Table 4). Of the four cancers with SMC2 frameshift mutations, none of them showed positive SMC2 immunostaining, and there was a significant difference of SMC2 expression between MSI-H cancers harboring mutations in SMC2 mutations and those with MSI-H that have not acquired mutations in SMC2 (Fisher’s exact test, p = 0.014). There were significant differences in SMC2 immunopositivity between the GC with MSI-H and the GC with MSI-L/MSS (Fisher’s exact test, p = 0.025), and between the CRC with MSI-H and the CRC with MSI-L/MSS (Fisher’s exact test, p = 0.012; Table 4). The immunostaining 502
Table 4. Summary of the SMC2 expression in gastric and colorectal cancers Positive SMC2 expression (%) GC with MSI-H (n = 32) 21 (65.6) CRC with MSI-H (n = 43) 27 (62.8) GC with MSI-L/MSS (n = 59) 50 (84.7) CRC with MSI-L/MSS (n = 60) 50 (83.3) MSI-H GC and CRC with 0 (0) SMC2 mutation (n = 4) MSI-H GC and CRC without 48 (67.6) SMC2 mutation (n = 71)
of SMC2, when present, was observed mainly in nuclei and less weakly in cytoplasm.
DISCUSSION Despite the importance of condensin functions in maintaining genome instability (2), genetic alterations of genes encoding components in condensin complexes are rarely known in human cancers. Based on the earlier report that showed SMC2 gene mutations in lymphomas with chromosomal destabilization (5), we attempted to address whether somatic frameshift mutation of SMC2 was present in GC and CRC tissues. We found that the mononucleotide repeats in coding region of SMC2 harbored frameshift mutations in both GC and CRC. The mutations were detected © 2014 APMIS. Published by John Wiley & Sons Ltd
SMC2 MUTATIONS IN GASTRIC AND COLORECTAL CANCERS
exclusively in the cancers with MSI-H, but not in those with MSI-L or MSS, indicating SMC2 gene is altered in GC and CRC with MSI-H by somatic frameshift mutations. In the present study, we detected two types of SMC2 mutations, both of which were novel mutations. Both mutations would cause change or delete amino acids after the frameshift mutation sites and hence resemble a typical loss-of-function mutation. The mutations at exon 6 and exon 17 would remove amino acids after the 188th residue and the 751th residues, respectively (Table 3). SMC2 protein is 1 198 amino acid-long and consists of N-terminal ATPase domain, N-terminal coiled-coil domain, hinge domain, C-terminal coiled-coil domain and C-terminal ATPase domain. The mutation at exon 6 would remove N-terminal coiled-coil domain, hinge domain, C-terminal coiled-coil domain and C-terminal ATPase domain, while the mutation at exon 17 would remove C-terminal coiled-coil domain and C-terminal ATPase domain. In cohesion I and II, SMC2 binds other SMC proteins and forms heterodimer for their functions (1, 2). It is likely that the deleted mutants would impair heterodimer formation and hamper its functions in maintaining chromosomal stability. We observed that SMC2 immunostaining was very weakly detectable or not detectable in all of the cancers with SMC2 frameshift mutations (Table 4). Because the antibody against SMC2 had been raised by a peptide immunogen within amino acid residues encoded by exon 27 that would be removed by the SMC2 frameshift mutations detected in the present study, the mutated SMC2 proteins might not be detected in cancer cells by this antibody. SSCP and direct sequencing of SMC2 in the mutated cases displayed that both of the frameshift mutations were heterozygous ones, suggesting that one allele was intact (Fig. 1). Loss of the SMC2 immunostaining in the cancers might be caused by the mutation in one allele and by the other gene silencing mechanisms in the second allele. Another possibility could be that quantity of SMC2 expression from one allele might not be enough to be detected in the immunohistochemistry. Of note, about one-third (23/71) of tumors even without SMC2 mutation showed loss of SMC2 expression by the immunohistochemistry (Table 4). These data suggest a possibility that loss of SMC2 expression in GC and CRC may be affected not only by frameshift mutations in the repeats, but also by other mechanisms such as epigenetic mechanisms or other somatic mutations outside of the frameshift mutation sites. Although condensin is important in maintaining chromosome stability, its implication in tumori-
© 2014 APMIS. Published by John Wiley & Sons Ltd
genesis has largely been unknown. An earlier study described that SMC2 was overexpressed in about 70% of CRC and the remaining 30% CRC were devoid of SMC2 expression (16). This report was in agreement with our data that showed loss of SMC2 expression in both CRC and GC. Functionally, depletion of SMC2 altered chromosome structure (2, 5). However, overexpression of C-terminus of SMC2 also disturbed chromosome condensation and mitotic progression (17). It should be studied in future whether overexpression or loss of expression or both of SMC2 is widespread in human cancers and whether consequences of each alteration are related to clinical phenotypes of cancers. Also, it remains to be clarified how the functions of SMC2 are different between cancers with MSI and without MSI, and what is the consequence of its frameshift mutations in their pathogenesis. In summary, our data indicate that SMC2 gene is altered in GC and CRC with MSI by frameshift mutation and loss of expression, and suggest that these alterations might contribute to pathogenesis of the cancers with MSI.
This work was supported by funding from Korea Research Foundation (2012R1A5A2047939).
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