Showing papers by "Nam Jin Yoo published in 2016"

BPTF, a chromatin remodeling-related gene, exhibits frameshift mutations in gastric and colorectal cancers.

Abstract: To the Editor Chromatin remodeling is a dynamic modification of chromatin architecture to allow access of the regulatory transcription proteins to condensed DNA, and thereby control gene expression. In addition to gene expression regulation, chromatin remodeling plays an epigenetic regulatory role in many processes, including DNA repair, cell death, cell cycle, and pluripotency, many of which are related to cancer development (1). Alterations in chromatin-remodeling genes, including ARID1A, MLL2, and ATRX, are found in many cancers (2). Chromatin remodeling is carried out by histone modifications and ATP-dependent chromatin remodeling complexes (1). The chromatin remodelers are subdivided into four families: SWI/SNF, INO80/SWR1, ISWI, and CHD families (1). BPTF (also known as NURF301) is an ISWI-containing ATP-dependent chromatin remodeling factor that mediates a direct preferential association with H3K4me3 tails (3). The essential role of BPTF in embryo development is well known (3), but its role in cancer development remains elusive. Many cancers, including bladder cancers and melanomas harbor somatic mutations of BPTF (4, 5) that are comprised of not only missense mutations but also truncating mutations (5). In a public genome database (http://genome. cse.ucsc.edu/), we found that BPTF gene have mononucleotide repeats in their coding sequences that could be targets for frameshift mutation in cancers with microsatellite instability (MSI). Frameshift mutations of genes with mononucleotide repeats are features of gastric (GC) and colorectal cancers (CRC) with MSI (6). Frameshift mutations in BPTF might cause alterations of their functions and contribute to cancer pathogenesis, but it remains unknown whether it is mutated in GC and CRC with MSI. To see whether BPTF gene harbored frameshift mutations of the repeats in GC and CRC, we analyzed the A9 repeat in exon 8, A8 repeat in exon 10, and A7 repeat in exon 23 in 34 GC with high MSI (MSI-H), 45 microsatellitestable/low MSI (MSS/MSI-L), 79 CRCs with MSIH and 45 CRCs with MSS/MSI-L by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) assay as described previously (7). The MSI evaluation system used five mononucleotide repeats (BAT25, BAT26, NR-21, NR-24, and MONO-27), tumoral MSI status of which was characterized as: MSI-H, if two or more of these markers show instability, MSI-L, if one of the markers shows instability and MSS, if none of the markers shows instability (8). In cancer tissues, malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides by microdissection (7). Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts in the SSCP as described previously (7). On the SSCP, we found aberrantly migrating bands in eight cases of the A9, two cases of the A8, and one case of the A7 (Fig. 1 and Table 1). DNA from the patients’ normal tissues showed no shifts in SSCP, indicating the aberrant bands had risen somatically. DNA sequencing analysis confirmed that the aberrant bands represented BPTF somatic mutations, which consisted of frameshift mutations by deletion of one base (c.2976delA, c.3259delA, and c.7725delA) or deletion of two bases (c.2975_2976delAA) or duplication of one base (c.2976dupA) in the repeats (Table 1). All of the mutations detected were interpreted as heterozygous according to the SSCP and direct sequencing analyses (Fig. 1A,B). The mutations were detected in cancers with MSI-H, but not in those with MSS/MSI-L. There was a statistical difference in the frameshift mutation frequencies between the cancers with MSI-H (11/113) and MSS/MSI-L (0/90) (Fisher’s exact test, p = 0.001). In terms of tissue origins, there was no statistical difference in mutation frequencies between GCs (2.94%, 1/34) and CRCs (12.7%, 10/79) with MSI-H (Fisher’s exact test, p = 0.084). There was no significant association of the mutations with the clinicopathologic data of the patient (age, sex, histologic grade, and stage). It is now well known that alteration of chromatin remodeling is actively involved in cancer development (1, 4, 5). Based on this, we attempted to disclose whether somatic frameshift mutations of a chromatin-remodeling gene BPTF were present in GC and CRC. We found that 2.9% of GCs and 12.7% of CRCs with MSI-H harbored BPTF frameshift mutations, indicating that BPTF gene is altered in GC and CRC with MSI-H by somatic frameshift mutation that might alter the function of BPTF protein. The mutations we found (Table 1) would delete amino acids after the frameshift mutations and hence would resemble a typical loss-of-function mutation. An earlier study identified that BPTF knockdown led

Frameshift mutations of a tumor suppressor gene ZNF292 in gastric and colorectal cancers with high microsatellite instability

Abstract: A transcription factor-encoding gene ZNF292 is considered a candidate tumor suppressor gene (TSG). Its mutations have been identified in cancers from liver, colon, and bone marrow. However, ZNF292 inactivating mutations that might suppress the TSG functions have not been reported in gastric (GC) and colorectal cancers (CRC) with microsatellite instability (MSI). In a public database, we found that ZNF292 gene had mononucleotide repeats in the coding sequences that might be mutation targets in the cancers with MSI. In this study, we analyzed 79 GCs and 124 CRCs including high MSI (MSI-H) and microsatellite stable/low MSI (MSS/MSI-L) cases for the detection of somatic mutations in the repeats. Overall, we identified frameshift mutations of ZNF292 in 3 (8.8%) GCs and 11 (13.9%) CRCs with MSI-H (14/113), but not in MSS/MSI-L cancers (0/90) (p < 0.001). Also, we studied intratumoral heterogeneity (ITH) of the ZNF292 frameshift mutations in 16 CRCs and found that two (12.5%) had regional ITH of the mutations. Our data show that ZNF292 gene harbors not only frameshift mutations but also mutational ITH, which together may be features of GC and CRC with MSI-H. Based on this, the ZNF292 frameshift mutations may possibly contribute to tumorigenesis by altering its TSG functions in GC and CRC.

Frameshift Mutations of AKAP9 Gene in Gastric and Colorectal Cancers with High Microsatellite Instability

Abstract: A-kinase-anchoring protein 9 (AKAP9) coordinates the cellular location and function of protein kinase A. AKAP9 plays an important role in centrosome duplication, cell cycle progression and maintenance of cell membrane integrity, alterations of which contribute to tumorigenesis. Somatic mutations of AKAP9 gene have been detected in many cancers including gastric (GC) and colorectal cancers (CRC), but the mutation status with respect to microsatellite instability (MSI) has not been reported. In a public database, we found that AKAP9 gene had mononucleotide repeats in the coding sequences that might be mutation targets in the cancers with MSI. We analyzed the mutations in 79 GCs and 124 CRCs including high MSI (MSI-H) and microsatellite stable/low MSI (MSS/MSI-L) cases by single-strand conformation polymorphism analysis and DNA sequencing. Overall, we found AKAP9 frameshift mutations in 4 (11.7 %) GCs and 20 (17.7 %) CRCs with MSI-H (24/113), but not in MSS/MSI-L cancers (0/90) (p < 0.001). In addition, we analyzed intratumoral heterogeneity (ITH) of AKAP9 frameshift mutations in 16 CRCs and found that five CRCs (31.3 %) harbored regional ITH of the AKAP9 frameshift mutations. Our data indicate that AKAP9 gene harbors not only somatic frameshift mutations but also mutational ITH, which together may be features of GC and CRC with MSI-H. Our results also suggest that regional mutation analysis is needed for a better evaluation of mutation status in these tumors to overcome ITH.

Putative Tumor Suppressor Genes EGR1 and BRSK1 Are Mutated in Gastric and Colorectal Cancers.

Abstract: Objective: The transcription factor-encoding EGR1 and the kinase-encoding BRSK1 are considered putative tumor suppressor genes (TSGs). However, EGR1 and BRSK1 mutations that could inactivate their functions are not reported in colorectal (CRC) and gastric (GC) cancers. Methods: There are mononucleotide repeats in EGR1 and BRSK1, which could be mutated in cancers with defects in mismatch repair, resulting in microsatellite instability (MSI). We analyzed 124 CRCs and 79 GCs for mutations and their intratumoral heterogeneities (ITHs). Results: Twenty-one out of 79 CRCs (26.6%) and 5 out of 34 GCs (14.7%) carrying high MSI (MSI-H) exhibited frameshift mutations. However, we found no such mutations in cancers with microsatellite stability. In addition, we studied ITH for these mutations in 16 cases of CRCs and observed that EGR1 and BRSK1 mutations exhibited ITH in 3 (18.8%) and 2 (12.5%) cases, respectively. Conclusion: Our data in this study reveal that the TSG genes EGR1 and BRSK1 carry mutational ITH as well as frameshift mutations in MSI-H CRC and GC, which together may be features of GC and CRC with MSI-H. These results suggest that frameshift mutations of EGR1 and BRSK1 might play a role in tumorigenesis through TSG inactivation in CRC and GC.

Frameshift Mutations of HSPA4 and MED13 in Gastric and Colorectal Cancers

Abstract: Frameshift mutation of genes containing mononucleotide repeats is a feature of gastric (GC) and colorectal cancers (CRC) with microsatellite instability (MSI). In the public genome database, we found that human HSPA4 gene encoding a heats hock protein 70 protein (HSP70–4) and MED13 gene had mononucleotide repeats in the coding sequences that could be targets for frameshift mutation in cancers with MSI. HSP70–4 is a member of HSP70 that is known to play a role in cell survival. MED13 is a member of MED genome-wide transcription regulators that function as a regulator for diverse biological processes. In this study, we analyzed the mutations in 79 GCs and 124 CRCs including high MSI (MSI-H) and microsatellite stable/low MSI (MSS/MSI-L) cases by single-strand conformation polymorphism analysis and DNA sequencing. We found frameshift mutations of HSPA4 gene in two cancers (one GC and one CRC) and MED13 gene in the other two cancers (one GC and one CRC). The frameshift mutations were deletions of one base (c.2396delA (p.Asn799MetfsX50)) in HSPA4 and (c.2175delA (p.Lys725AsnfsX4)) in MED13. Each of HSPA4 and MED13 mutations were detected in GC with MSI-H (1/34: 2.9 %) and CRC with MSI-H (1/79: 1.3 %), but not in those with MSS. Our data show that unconventional HSPA4 and MED13 genes harbored frameshift mutations in GC and CRC with MSI. These mutations might possibly inactivate their functions and could be a feature of GC and CRC with MSI-H.

Frameshift Mutation of ASPM Gene in Colorectal Cancers with Regional Heterogeneity

Abstract: To the Editor: Recent advance in genome sequencing technologies such as whole-exome sequencing has provided huge data sets of somatic mutations in major cancer types [1]. The Pan-Cancer project integrated overall genome sequencing data, subsequently identified mutations across the cancers, found their organ specificity and identified molecular commonalities across tumor types [1, 2]. During the Pan-Cancer efforts, many of the mutations that occurred at a low frequency in a cancer type were shared by sets of cancer types and were predicted as significantly mutated genes [1–3]. One of such genes is ASPM that is the human ortholog of the Drosophila melanogaster abnormal spindle gene, which is essential for normal mitotic spindle function in the cells [4]. Germline mutations in ASPM are associated with microcephaly primary type 5 [4]. ASPM was initially identified as a protein that regulated neurogenesis but it was later known to be widely expressed in a variety of normal and malignant tissues [5]. ASPM is overexpressed in many cancers and considered a poor prognostic marker [6]. ASPM mutations have been identified in several cancers with a low prevalence, but it became a significantly mutated gene by the Pan-Cancer analysis [3]. Colorectal cancer (CRC) and gastric cancer (GC) harbored ASPM mutation in 4.35 % and 1.31 %, respectively [3], all of which were missense or splicing-site mutations (search at www.intogen.org). Through a search in the UCSC public genome database (http://genome.cse.ucsc.edu/), we found that the ASPM gene harbors a mononucleotide repeat in the coding sequence that might serve as a target for mutation in GC and CRCs exhibiting microsatellite instability (MSI) [6]. To see whether ASPM gene harbored frameshift mutations of the repeat in GC and CRC, we analyzed the exon 18 A7 repeats in 34 GC with high MSI (MSI-H), 45 GC with stable MSI/low MSI (MSS/MSI-L), 76 CRC with MSI-H and 45 CRC with MSS/MSI-L by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) assay as described previously [7]. In cancer tissues, malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides by microdissection [7, 8]. Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts in the SSCP as described previously [7, 8]. In addition, to see whether frameshift mutations in the ASPM gene repeat possess intra-tumor heterogeneity (ITH) that contributes to tumor aggressiveness [9], we analyzed 16 CRCs with four to seven regional biopsies per CRC. On the SSCP, we observed aberrant SSCP bands of ASPM gene in three CRCs and two GCs. DNA from the patients’ normal tissues showed no shifts in SSCP, indicating the aberrant bands had risen somatically. DNA sequencing analysis confirmed that the aberrant bands Eun Ji Choi and Min Sung Kim contributed equally to this work.

Absence of KNSTRN Mutation, a Cutaneous Squamous Carcinoma-Specific Mutation, in Other Solid Tumors and Leukemias.

Abstract: To the Editor: KNSTRN gene encodes the kinetochore-localized astrin/ spag5-binding protein that modulates chromosome segregation during mitosis [1]. A recent genomic study identified recurrent somatic mutations of the KNSTRN in 19 % of cutaneous squamous cell carcinoma (SCC) [2]. Of the mutations detected, more than half of them were recurrent at a specific residue (p.Ser24Phe). In addition, the p.Ser24Phe mutation was found in other cutaneous tumors such as malignant melanomas (2 %) and actinic keratosis (19 %) [2]. Functionally, the KNSTRN p.Ser24Phe mutation disrupted chromatid cohesion in normal cells, correlated with increased aneuploidy in primary tumors and enhanced tumorigenesis in vivo [2]. In other reports, aberrant KNSTRN expression was shown to result in loss of chromatid cohesion in a non-cutaneous tumor cell line HeLa cells [1]. Also, KNSTRN is expressed in a broad range of normal human tissues [2]. These data suggest a possibility that alterations of KNSTRN gene might be present not only in skin but also in other tissues. Since the KNSTRN p.Ser24Phe mutation is considered a driver mutation with a high recurrence, it may be interesting to know whether the mutation occurs in other human tumors besides cutaneous tumors. For this, tumor tissues from 2229Korean patients, including hematologic, epithelial and mesenchymal tumor from various origins, were used for this study (Table 1). Prostate and ovarian tissues were fromKorea Prostate Bank andKoreaGynecologic Cancer Bank, respectively. The tumors did not include cutaneous SCC where KNSTRN p.Ser24Phe mutations were recurrent, because the cutaneous SCC tissues were not available in this study. For solid tumors, malignant and normal cells were selectively procured from by microdissection [3, 4]. Approval for this study was obtained from the institutional review board. We analyzed exon 1 of KNSTRN gene that encompassed p.Ser24Phe mutation sites by polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP). Genomic DNA each from tumor and normal cells was ampl i f i ed by PCR wi th a pr imer pa i r (5 ′ CTCTGAGCGAACCTTCCGTA-3′ (forward) and 5′CCGCCTGGGTTTCAAATAG-3′ (reverse); product size: 145 base pairs). Other procedures of the PCR-SSCP were described in our previous studies [3, 4]. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts. On the SSCP, all of the PCR products for KNSTRN exon 1 were clearly seen. However, none of the SSCP from the cancers displayed aberrantly migrating bands compared to wildtype bands from the normal tissues, indicating there was no evidence of KNSTRN exon 1 mutations in the tumors. To confirm the SSCP data, we repeated the experiments twice, including tissue microdissection, PCR and SSCP to ensure specificity of the results, and found that the data were consistent. An interesting point in cancer genetics is to identify whether any mutation found in a specific tumor type is common to other types. The present study, however, detected no somatic mutations of KNSTRN p.Ser24Phe * Sug Hyung Lee suhulee@catholic.ac.kr

Inactivating Frameshift Mutation of INPP4B Encoding a PI3K Pathway Phosphatase in Gastric and Colorectal Cancers.

Abstract: To the Editor: The PI3K-AKT pathway regulates crucial cellular processes such as cell proliferation and survival, and is frequently altered in cancers. PIK3CA (lipid kinase) and PTEN (lipid phosphatase) in this pathway are oncogene and tumor suppressor genes (TSGs), respectively [1]. Another lipid phosphatase INPP4B (inositol polyphosphate-4-phosphatase, type II) in this pathway suppressed PI3K–AKT pathway and exhibited tumorsuppressing activity in cells [1]. Decreased INPP4B expression was found in many cancers [2]. In vivo, INPP4B suppressed development and metastasis of thyroid tumors [3]. Together, these data strongly suggest that INPP4B has TSG functions. However to date, it remains unknown whether somatic mutation of INPP4B are common in gastric cancer (GC) and colorectal cancer (CRC). In a public genome database (http://genome.cse.ucsc.edu/), we found that human INPP4B had a mononucleotide repeat that could be a target for frameshift mutation in GC and CRC with microsatellite instability (MSI) [4]. In this study, we analyzed an A7 repeat in the INPP4B exon 25 by polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) assay. We used methacarn-fixed tissues of 79 CRCs with high MSI (MSI-H), 53 microsatellite stable (MSS) CRCs, 34 GC with MSI-H and 45 GC with MSS. In cancer tissues, malignant cells and normal cells were selectively procured by microdissection [5]. Radioisotope ([P]dCTP) was incorporated into the PCR products, which were subsequently displayed in SSCP gels and analyzed with direct DNA sequencing [5]. On the SSCP, we observed aberrant bands of INPP4B gene in one GC and two CRCs. DNA from the patients’ normal tissues showed no shifts in SSCP compared to tumor DNA, indicating the aberrant bands had risen somatically. DNA sequencing analysis confirmed that the aberrant bands represented a recurrent PBRM1 somatic mutation, which was an identical frameshift mutation (deletion of one base) in the A7 repeat (c.2452delA) that would result in a frameshift mutation (p.Arg818GlufsX4) (Fig. 1). They were detected in two CRCs (2/79: 2.5 %) and one GC (1/34: 2.9 %) withMSI-H, but not in those with MSS (0/98). The frameshift mutation detected in the current study would result in a premature stop of amino acid synthesis in INPP4B protein and hence resembles a typical loss-offunction mutation. Based on the earlier data that showed TSG activities of INPP4B in cells [2, 3], the inactivating mutation found in this study could contribute to cancer development by inhibiting the TSG activities. Our data show that GC and CRC with MSI-H could harbor INPP4B truncation mutation that might alter PI3K pathway. Such data might provide useful information in evaluating lipid kinase-targeting drugs in cancer patients. * Sug Hyung Lee suhulee@catholic.ac.kr

Leukemia Relapse-Associated Mutation of NT5C2 Gene is Rare in de Novo Acute Leukemias and Solid Tumors.

Abstract: To the editor: The 5′-nucleotidase cytosolic II (NT5C2), also known as cN-II, encodes a hydrolase that plays an important role in cellular purine metabolism. It inactivates 6-thioinositol monophosphate (MP) and 6-thioguanosine MP, which mediate the cytotoxic effects of 6-MP and 6-thioguanine (6-TG) that are used in the treatment of acute lymphoblastic leukemias (ALL) [1]. Recent studies discovered that somatic mutations of NT5C2 were common in relapsed T-ALL and less frequently in B-ALL [2, 3]. The NT5C2 mutations were recurrent in specific amino acids (most frequently in p.R367Q and p.R238W) and appeared gain-of-function mutations that enhanced the enzymatic activity, resulting in inactivation of 6MP and 6-TG [2, 3]. They also found that NT5C2 mutations had existed before the relapses as a rare clone [2], indicating that NT5C2 mutations exist at diagnosis in ALL and emerge after 6-MP is treated. In the COSMIC database, some solid cancers (gastric, colon and endometrial cancers) harbored the relapse-specific NT5C2 mutation p.R367Q, which emerged without any history of chemotherapy, suggesting either that the NT5C2 mutation had been raised by other factors besides chemotherapy or that it might play a role in development rather than relapse of the cancers. However, the status of NT5C2mutations remains unknown in primary tumors without exposure to 6-MP. A similar situation was also identified in the case of relapsespecific EGFR mutation p.T790M, which was also identified in lung cancers that had never been exposed to gefitinib therapy [4]. Thus, it is interesting to study whether the NT5C2mutations are present in primary hematologic neoplasia as well as in primary solid tumors. For this, we analyzed the NT5C2 somatic mutations using genomic DNA from in fresh bone marrow aspirates of 705 hematologic tumors (acute myelogenous leukemias (AML), ALL, multiple myelomas and myelodysplastic syndromes) (Table 1) by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) assay. Also, we analyzed the gene in paraffin-embedded tissues of 150 nonHodgkin lymphomas (NHL) and 1639 solid tumors (Table 1). Approval was obtained from the Catholic University of Korea, College of Medicine’s institutional review board for this study. Genomic DNA each from tumor cells and normal cells (remission bone marrow cells in the cases of leukemias) were used in this study. Because the relapse-specific mutations of NT5C2 have been detected in exons 11 and 15 [2, 3], we analyzed these two exons in this study by polymerase chain reaction (PCR)based single-strand conformation polymorphism (SSCP). Radioisotope was incorporated into the PCR products for detection by autoradiogram. Other procedures of the PCR-SSCP were described in our previous studies [4, 5]. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts. PCR and subsequent SSCP analysis detected aberrant migrating SSCP bands in two tumors (one AML and one colon cancer), but not in the other hematologic nor solid tumors (2/2496, 0.08 %). Direct DNA sequencing analyses for the two cases with aberrant bands led us to identify that the aberrant bands represented NT5C2 somatic mutations. The mutations were missense mutations that substituted two different amino acids (p.Glu240Gln in the AML and p.Arg363Gln in Hye Rim Oh and Youn Jin Choi contributed equally to this work.

Mutational and expressional alterations of ZMPSTE24, DNA damage response-related gene, in gastric and colorectal cancers

Abstract: Loss of ZMPSTE24 is related to progeroid phenotypes in human. Cells in zmpste24-deficient mice show delayed DNA damage response, increased aneuploidy and increased genomic instability, which are considered features of cancer cells. The aim of this study was to address whether ZMPSTE24 gene was mutated in colorectal cancers (CRCs) and gastric (GCs), and its expression was altered. ZMPSTE24 possesses a T9 mononucleotide repeat in an exon, which could be mutated in cancers with defects in mismatch repair that can result in microsatellite instability (MSI). For this, the current study studied 124 CRCs and 79 GCs for mutation and expression analyses. For mutations in the T9, CRCs (16.4%) and GCs (8.8%) with high MSI (MSI-H), but not microsatellite stable/low MSI (MSS/MSI-L), exhibited frameshift mutations. Also, the ZMPSTE24 mutations showed intratumoral heterogeneity (ITH) in 4 of 16 CRC cases. Downregulation of ZMPSTE24 protein expression was found in 16.9% of CRCs and 8.9% of GCs by immunohistochemistry. Our study found frameshift mutation and its ITH in ZMPSTE24 gene as well as downregulation of ZMPSTE24 expression. Based on these observations, the present study suggests that inhibition of ZMPSTE24 by both mutational and expressional pathways might together play a role in tumorigenesis of CRC and GC harboring MSI-H phenotype.

Frameshift Mutations of CAB39L, an Activator of LKB1 Tumor Suppressor, in Gastric and Colorectal Cancers

Abstract: To the Editor: Calcium binding protein 39-like (CAB39L) is a core component of LKB1 tumor suppressor complex and activates LKB1 [1]. Activation of LKB1 plays crucial roles maintaining cell polarity and loss of LKB1 leads to disorganization of cell polarity and facilitates tumor growth. Germline mutations in LKB1 are associated with Peutz-Jeghers syndrome characterized by polyps in the gastrointestinal tract and other neoplasms [2]. Somatic mutations of LKB1 gene were also found in many tumors [3]. Also, CAB39L binds and activates STK24, a STE20 family protein kinase [4]. STK24 not only inhibits cell cycle progression by phosphorylating NDR kinase, but also promotes cell death [5]. These data indicate that that CAB39L is possibly involved in cancer-related pathways and suggest that inactivation of CAB39L might be related to tumorigenesis. However, its implications in cancer development are not unknown. In a public genome database (http://genome.cse.ucsc.edu/), we found that human CAB39L had mononucleotide repeats in the coding sequences that could be targets for frameshift mutation in cancers with microsatellite instability (MSI). Frameshift mutation of genes containing mononucleotide repeats is a feature of gastric (GC) and colorectal cancers (CRC) withMSI [6]. To date, however, it is not known whether CAB39L gene is mutationally altered in GC and CRC. In this study, we analyzed an A7 repeat in theCAB39L exon 2 by polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) assay. For this, we used methacarn-fixed tissues of 34 GC with high MSI (MSI-H), 45 GC with stable MSI (MSS), 89 CRC with MSI-H and 45 CRCwithMSS. For 16 of the 89 CRCwithMSI, we collected four to seven different tumor areas from the same patients and analyzed intratumoral heterogeneity (ITH) of CAB39L mutation. In cancer tissues, malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides by microdissection [7]. Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts in the SSCP as described previously [8]. On the SSCP, we observed aberrant bands ofCAB39L gene in two cancers (a GC and a CRC). DNA from the patients’ normal tissues showed no shifts in SSCP, indicating the aberrant bands had risen somatically. DNA sequencing analysis confirmed that the aberrant bands representedCAB39L somatic mutations, which were a frameshift mutation by duplication of one base (c.10dupA (p.Met4AsnfsX5)) and another frameshift mutation by deletion of one base (c.10delA (p.Met4CysfsX15)) in the A7 repeat. They were detected in a GCwithMSI-H (1/34: 2.9%) and a CRCwithMSI-H (1/89: 1.1 %), but not in those with MSS. The mutational ITH analysis in the 16 CRCs (4–7 fragments per case) with MSI-H identified that the same CRC described above harbored ITH Mi Ryoung Choi and Chang Hyeok An contributed equally to this work.

ADNP encoding a transcription factor interacting with BAF complexes exhibits frameshift mutations in gastric and colorectal cancers

Abstract: Sir,Somatic mutations of genes for gene components for chromatin remodeling machineries are frequent targets in human cancers, for example, ARID1A gene [1] that is a component of BAF complex involv...

Absence of PRKD1 Mutation, a Salivary Tumor-Specific Mutation, in Solid Tumors and Leukemias.

Abstract: To the Editor: A recent genomic study showed that somatic mutations of a serine/threonine kinase-encoding gene PRKD1 were very common in polymorphous low-grade adenocarcinoma (PLGA) of salivary gland [1]. They found that PRKD1 hotspot mutations encoding p.Glu710Asp were recurrent in 72.9 % of PLGAs but not in other salivary gland tumors. PRKD1 plays a role in cell adhesion, migration, vesicle transport and survival [2]. Functional studies demonstrated that this kinase-activating mutations altered glandular structures into larger, coalescent structures with filled lumens and irregular contours not uncommonly displaying infiltrating edges, a phenotype consistent with that induced by the forced expression of other oncogenes in this model system [3]. Because PRKD1 p.Glu710Asp mutations are considered driver mutations and highly recurrent, it may be interesting to know whether the PRKD1 p.Glu710Asp mutations occur in other human tumors besides PLGA. For this, tumor tissues from 2444 Korean patients, including hematologic, epithelial and mesenchymal tumor from various origins, were used for this study (Table 1). The tumors did not include PLGAs where PRKD1 p.Glu710Asp mutations are recurrent, because PLGA tissues were not available in this study. For solid tumors, malignant and normal cells were selectively procured from by microdissection [4]. Approval for this study was obtained from the institutional review board. We analyzed exon 10 of PRKD1 gene that encompassed p.Glu710Asp mutation sites by polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP). Genomic DNA each from tumor and normal cells was ampl i f i ed by PCR wi th a pr imer pa i r (5 ′ AGGTTTTAGATGCCACAAAG-3′ (forward) and 5′CCAGCTTACATTGCCATAG-3′ (reverse); product size: 185 base pairs). Other procedures of the PCR-SSCP were described in our previous studies [5]. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts. On the SSCP, all of the PCR products for PRKD1 exon 10 were clearly seen. However, none of the SSCP from the cancers displayed aberrantly migrating bands compared to wildtype bands from the normal tissues, indicating there was no evidence of PRKD1 exon 10 mutations in the tumors. To confirm the SSCP data, we repeated the experiments twice, including tissue microdissection, PCR and SSCP to ensure specificity of the results, and found that the data were consistent. One of the main concerns in cancer genetics is to identify whether anymutation found in a tumor is common in the other tumor types. Based on the earlier data that PRKD1 mutation was recurrent in PLGAs [1], we attempted to determine whether somatic mutation of the recurrent site was present in other tumors in this study. The present study, however, detected no somatic mutations of PRKD1 p.Glu710Asp in 2444 tumors from 24 types. Our data indicate that the PRKD1 p.Glu710Aspmay be specific to PLGA, but not to other tumor development. The discovery of the recurrent PRKD1 mutations offered an opportunity for developing therapeutic and diagnostic tools targeting the mutations in tumors. Our data, however, suggest that such applications mutations should be limited to PLGA tumors.

Inactivating frameshift mutation of PBRM1, a putative tumour suppressor gene, in colorectal cancers.

Abstract: Chromatin remodelling controls not only gene expression but also plays important roles in DNA repair, cell death and cell cycle, which are related to cancers.[1] Alterations in chromatin-remodelling genes, including ARID1A, MLL2 and ATRX, are found in many cancers.[2,3] The chromatin remodellers are subdivided into four families: SWI/SNF, INO80/SWR1, ISWI and CHD families.[1] PBRM1, a member of SWI/SNF family, encodes a subunit of ATP-dependent chromatin-remodelling complexes.[1] Renal cell carcinoma, transitional cell carcinoma and cholangiocarcinoma harbour frequent somatic mutations of PBRM1 gene, many of which are inactivating mutations.[3–5] Functionally, suppression of PBRM1 expression promoted cell proliferation and cell cycle progression,[4,6] indicating that PBRM1 is a candidate tumour suppressor gene (TSG). However to date, it remains unknown whether PBRM1 inactivating mutations are common in colorectal cancer (CRC). In a public genome database (http://genome.cse. ucsc.edu/), we found that human PBRM1 had a mononucleotide repeat in the coding sequence that could be a target for frameshift mutation in cancers with microsatellite instability (MSI). Frameshift mutation of genes containing mononucleotide repeats is a feature of CRC with MSI.[7] In this study, we analysed an A7 repeat in the PBRM1 exon 9 by polymerase chain reaction (PCR)based single strand conformation polymorphism (SSCP) assay. We used methacarn-fixed tissues of 79 CRCs with high MSI (MSI-H) and 53 microsatellite stable (MSS) CRCs. In cancer tissues, malignant cells and normal cells were selectively procured from haematoxylin and eosin-stained slides by microdissection.[8] Radioisotope

Frameshift Mutation of MED25, a Transcription Regulator, and its Mutational Heterogeneity in Colorectal Cancers

Abstract: To the Editor: MED12 somatic mutations are highly recurrent in uterine leiomyoma and breast fibroadenoma, suggesting thatMED gene mutations might play roles in tumor development [1, 2]. The Mediator (MED) functions as a bridge to convey information from gene-specific regulatory proteins to the basal RNA polymerase II transcription machinery [3]. Mediator is recruited to promoters by direct interactions with regulatory proteins and serves as a scaffold for the assembly of a functional preinitiation complex with RNA polymerase II and the general transcription factors. There are more than 20 MED complexes that exist in two distinct forms, i.e., CDK8-mediators and non-CDK8 core mediators [3]. In addition to the roles in general transcription, the MEDs function as a regulator for diverse biological processes, including differentiation, proliferation and tumorigenesis that are related to tumor development [3]. MED25 (also known as ACID1, ARC92 and PTOV2), a non-CDK8 coreMED, is functionally associated with the activation domains of multiple cellular and viral transcriptional activators, including the herpes simplex viral activator VP16, sterol regulatory element binding protein and NF-kappa B [4]. Transcriptional activity of RA receptor that plays a role in cancer therapy is enhanced by association of MED25 with CREB-binding protein [4]. However, alterations of MED25 in cancers remain unknown. Cancer development initiates through a clonal expansion of a single cell, but the cells usually become heterogeneous after branching subclonal expansions, which leads to intra-tumor heterogeneity (ITH). This ITH contributes to tumor aggressiveness and may impede the accurate diagnosis/prognosis [5]. In a public genome database (http://genome.cse.ucsc.edu/), we found that human MED25 had a mononucleotide repeat in the coding sequences that could be a target for frameshift mutation in cancers exhibiting microsatellite instability (MSI). Frameshift mutation of genes containing mononucleotide repeats is a feature of colorectal cancers (CRC) with MSI [6]. To date, however, it is not known whether MED25 gene is mutationally altered in CRC with MSI. In this study, we analyzed a C7 repeat in the MED25 exon 6 by polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) assay. We used methacarn-fixed tissues of 89 high MSI (MSI-H) CRCs and 52 microsatellite-stable (MSS) CRCs. For 16 of the 89 MSI-H CRCs, we collected four to seven different tumor areas from the same patients and analyzed ITH ofMED25mutation. In cancer tissues, malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides by microdissection [7]. Radioisotope ([P] dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts in the SSCP as described previously [7]. On the SSCP, we observed aberrant bands ofMED25 gene in seven CRCs. DNA from the patients’ normal tissues showed no shifts in SSCP, indicating the aberrant bands had risen somatically. DNA sequencing analysis confirmed that the aberrant bands represented a recurrent MED25 frameshift * Sug Hyung Lee suhulee@catholic.ac.kr