Showing papers by "Sug Hyung Lee published in 2013"

Mutational and expressional analyses of SPOP, a candidate tumor suppressor gene, in prostate, gastric and colorectal cancers.

Abstract: Mounting evidence exists that alterations of ubiquitination processes are involved in cancer pathogenesis. Speckle-type POZ protein (SPOP) is a key adaptor for Cul3-based ubiquitination process. Recent studies reported that SPOP may be a tumor suppressor gene (TSG) and somatic mutation of SPOP was detected in prostate cancer (PCA). The aim of this study was to see whether alterations of SPOP protein expression and somatic mutation of SPOP gene are features of cancers. In this study, we analyzed SPOP somatic mutation in 45 gastric (GC), 45 colorectal cancer (CRC) and 45 PCA by single-strand conformation polymorphism (SSCP). Also, we analyzed SPOP protein expression in 60 GC, 60 CRC and 60 PCA by immunohistochemistry. Overall, we detected three somatic missense mutations of SPOP gene in the coding sequences (p.Ser14Leu, p.Tyr87Cys and p.Phe133Leu). The mutations were observed in two PCA and one CRC. Of note, the p.Phe133Leu was a recurrent mutation reported in an earlier study. In the immunohistochemistry, SPOP protein was expressed in normal gastric, colonic and prostate epithelial cells, whereas it was lost in 30% of GC, 20% of CRC and 37% of PCA. Our data indicate that loss of SPOP expression was common in GC, CRC and PCA, but somatic mutation of SPOP in this study was rare in these tumors. Also, the data provide a possibility that loss of expression of SPOP gene might play a role in cancer pathogenesis by altering TSG functions of SPOP.

Mutational analysis of splicing machinery genes SF3B1, U2AF1 and SRSF2 in myelodysplasia and other common tumors.

Abstract: Recurrent somatic mutations in splicing machinery components, including SF3B1, U2AF1 and SRSF2 genes have recently been reported in myelodysplastic syndromes (MDS). Such a recurrent nature strongly suggests that these mutations play important roles in tumor development. To see whether SF3B1, U2AF1 and SRSF2 mutations occur in other human tumors besides MDS, we analyzed the hotspot mutation regions of these genes in 2,345 tumor tissues from various origins (61 MDS, other 616 hematologic tumors, 1,421 epithelial tumors and 247 non-epithelial stromal tumors) by single-strand conformation polymorphism analysis. We found SF3B1, U2AF1 and SRSF2 mutations in 5 (8.2%), 12 (19.7%) and 8 (13.1%) of 61 MDS, respectively. We also confirmed these mutations in other myeloid neoplasia, including de novo acute myelogenous leukemia (AML), chronic myelomonocytic leukemia and MDS/myeloproliferative disorder. In addition, we discovered that the SRSF2 gene was mutated in two childhood acute lymphoblastic leukemias (childhood ALL) (1.5%). In solid tumors, we found SF3B1 mutations in gastric and prostate cancers, and U2AF1 mutation in a borderline mucinous tumor of ovary, but the overall incidences of the hotspot mutation regions were very low (0.2%). Our data suggest that SF3B1, U2AF1 and SRSF2 mutations occur not only in myeloid lineage tumors but also in lymphoid lineage tumors. The data suggest that the splicing gene mutations play important roles in the pathogenesis of hematologic tumors, but rarely in solid tumors.

Mutational analysis of DNMT3A gene in acute leukemias and common solid cancers.

Abstract: DNMT3A, a DNA methyltransferase that functions for de novo methylation, is important in development and many cellular processes related to tumorigenesis. Somatic mutations of DNMT3A gene, including recurrent mutations in its Arg-882, were recently reported in acute myelogenous leukemia (AML), strongly suggesting its role in development of AML. To see whether DNMT3A mutation occurs in other malignancies as well, we analyzed DNMT3A in 916 cancer tissues from 401 hematologic malignancies (AML, acute lymphoblastic leukemias (ALL), multiple myelomas and lymphomas) and 515 carcinomas (lung, breast, prostate, colorectal and gastric carcinomas) using a single-strand conformation polymorphism (SSCP) assay. We identified DNMT3A mutations, especially the Arg-882 mutations, in adulthood AML (9.4%). In addition, we found DNMT3A mutations in pre-B-ALL and three lung cancers at lower frequencies. Allelic loss of DNMT3A was frequently observed in most cancer types analyzed, including lymphomas (48.1%), gastric cancers (23.5%) and lung cancers (18.3%) irrespective of DNMT3A mutation. Also, loss of DNMT3A expression was common in lung cancers (46.4%), and was associated with the allelic loss. Our data indicate that DNMT3A gene is mutated mainly in AML, but it occurs in other cancers, such as ALL and lung cancer, despite the lower incidences. Also, the data suggest that DNMT3A is altered in many cancer types by various ways, including somatic mutations, allelic loss and loss of expression that might play roles in tumorigenesis.

DICER1 exons 25 and 26 mutations are rare in common human tumours besides Sertoli–Leydig cell tumour

Abstract: approach, a 3% increase in microscopic involvement of the bladder neck and a 2% increase in microscopic involvement of the seminal vesicle have been seen. Our approach has not helped us to solve the problem of a true intraprostatic component of the seminal vesicles in addition to the anatomically well-characterized extraprostatic seminal vesicle. The seminal vesicle connects with the prostate ejaculatory duct, and occasionally appears to be invaginated into the prostate (Figure 6). In conclusion, our current approach could contribute to more accurate evaluation of the prognostic morphological elements included by Srigley and his co-authors in their dataset for reporting of prostate carcinoma in RPSs, and also to meaningful comparisons of benchmarking data, epidemiological studies, and clinical trials.

Frameshift mutations of a chromatin‐remodeling gene SMARCC2 in gastric and colorectal cancers with microsatellite instability

Abstract: To the Editor Cellular machineries that allow transcription factors to access target promoters are largely divided into two: the chromatin-remodeling complex and the histone acetylases (1). In yeast, the multisubunit SWI/SNF complex is thought to be responsible for chromatin remodeling. The SWI/SNF family proteins display helicase and ATPase activities that are thought to regulate transcription of certain genes by altering chromatin structures (2). The SWI-SNF complex regulates gene expressions and plays important roles in development, differentiation, and proliferation of cells (2). Also, the complex is involved in the pathogenesis of cancer. For example, ARID1A encoding a component of the SWI-SNF complex, is considered a tumor suppressor gene and is mutated in many cancers (3–5). SWI/SNFrelated, matrix-associated, actin-dependent regulator of chromatin, subfamily c (SMARC) proteins comprise parts of SNF/SWI complex (1). In contrast to the ARID1A, cancer-related functions of which are well known, those of SMARC genes remain unknown. By analyzing human gene sequences in a public database (http://genome.cse.ucsc.edu/), we found that SMARCB1 (A7 in exon 2), SMARCC1 (A7 in exon 23) and SMARCC2 (C8 in exon 26) genes have mononucleotide repeats in the coding sequences that could be targets for frameshift mutation in the cancers with microsatellite instability (MSI). About 10 –30% of in gastric (GC) and colorectal (CRC) cancers with defective DNA mismatch repair (MMR) show microsatellite instability (MSI). To see whether SMARCB1, SMARCC1, and SMARCC2 genes are involved in cancer development, we analyzed their mononucleotide repeats in methacarn-fixed tissues of 46 GC and 56 CRC with MSI by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) assay as described previously (6, 7). We used 32 GC with high MSI (MSI-H), 14 GC with low MSI (MSI-L), 41 CRC with MSI-H, and 15 CRC with MSI-L. Malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle by microdissection as described

NIPBL, a Cohesion Loading Factor, Is Somatically Mutated in Gastric and Colorectal Cancers with High Microsatellite Instability

Abstract: Timely segregation of sister chromatids during the cell cycle is essential for maintaining euploidy [1]. Aberrant segregation into daughter cells promotes aneuploidy and chromosome instability, which are features of many tumors [1]. Chromatid cohesion, a physical linkage of replicated sister chromatids, is mediated by a multiprotein complex called cohesin [1]. NIPBL is a cohesion loading factor that is required for cohesin to interact with chromosomes [2, 3]. NIPBL mutations (missense, nonsense, frameshift and deletion mutations) account for approximately 60 % of Cornelia de Lange syndrome, a congenital disorder with multisystem developmental abnormalities [3]. In Cornelia de Lange syndrome, mutated NIPBL interferes with its interaction with its partners in the cohesion complex [3]. Components in the cohesin complex and cohesin regulatory proteins are frequently altered at expression and mutation levels in human cancers [1]. For example, cohesin protector SGOL1 down-regulation leads to chromosomal instability in colorectal cancers (CRC) [4]. Somatic mutations of cohesion regulatory factor PDS5B have been identified in prostate cancers [5]. These data suggest that cohesin-related genes could be considered potential tumor suppressor genes (TSG) [1]. Somatic mutations of the NIPBL gene have been found in CRC with stable miscrosatellite instability (MSI) that may underlie chromosomal instability in CRC [6]. Together, these data suggest a possibility that NIPBL is a candidate TSG. In a public genome database (http://genome.cse.ucsc. edu/), we found that the human NIPBL gene had mononucleotide repeats in its coding sequences that could be targets for frameshift mutation in cancers with high MSI (MSI-H) [7]. To data, however, the data on NIPBL somatic mutation in gastric (GC) and CRC with MSI-H are lacking. To see whether the mononucleotide repeats in the NIPBL gene are mutated in GC and CRC, we analyzed two A7 and one A8 repeats in NIPBL exon 10 by polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) assay with three primer pairs. For this, we used 32 GC with MSI-H, 59 GC with MSS, 47 CRC with MSI-H and 53 CRC with MSS in methacarnfixed tissues. All of the patients of the cancers were Koreans. In cancer tissues, malignant cells and normal cells were selectively procured from hematoxylin and eosinstained slides by microdissection [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. On the SSCP, we observed aberrant bands of the NIPBL gene in 11 cancers (Fig. 1). DNA from normal tissue Min Sung Kim and Chang Hyeok An contributed equally to this study.

Somatic Mutation of PARK2 Tumor Suppressor Gene is not Common in Common Solid Cancers

Abstract: Recent studies identified that PARK2 gene was a candidate tumor suppressor gene in colorectal cancers and glioblastomas. The aim of this study was identify whether PARK2 somatic mutation is present in other solid tumor as well. In this study, we analyzed the entire coding sequences of human PARK2 gene in gastric, colorectal, breast, lung and prostate carcinoma by single-strand conformation polymorphism (SSCP) and subsequent direct DNA sequencing. We found two missense mutations (p.Ser9Thr and p.Gly450Val) in colon carcinomas (4.3 %), which were not overlapped with the known PARK2 mutations. Our data suggest that somatic mutational events in PARK2 gene may be rare in colorectal, gastric, prostate, breast and lung carcinomas and may not play an important role in the development of these cancers.

Somatic mutation of H3F3A, a chromatin remodeling gene, is rare in acute leukemias and non-Hodgkin lymphoma.

Abstract: To the Editor: Chromatin remodeling is a dynamic modification of chromatin architecture to allow access of genomic DNA to the regulatory transcription machineries, and thereby control gene expression (1). Chromatin remodeling provides wellorchestrated regulation of crucial cell growth and division steps and therefore involved in tumorigenesis (1). Histones are chief components of chromatin, acting as spools around which DNA winds, and play a role in gene regulation. Histones H2A, H2B, H3, and H4 are known as core histones, while histones H1 and H5 are known as the linker histones. Recent studies found that the gene encoding H3 histone family 3A (H3F3A) was somatically mutated in glioblastoma (2–4). Of note, such mutations were recurrent in two amino acid residues (p.K27M, p.G34R/p.G34V) within the histone tails. Mutations of chromatin remodeling genes, including ATRX, UTX, MLL, EP300, ARID1A, and CHD6, have been also identified in solid cancers (5–7). Thus, it is interesting to know whether H3F3A mutation occurs in tumors from hematopoietic system as well. However, to date, the data on H3F3A somatic mutation in other human tumors besides glioblastomas are lacking. For this, we analyzed somatic mutations of H3F3A gene in fresh bone marrow aspirates of 911 hematologic tumors (acute myelogenous leukemias (AML), acute lymphoblastic leukemias (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 93 non-Hodgkin lymphomas (NHL). 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. For NHL, malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides by microdissection as described previously (8). Up to now, all of the H3F3A somatic mutations have been detected at nucleotide sequences encoding amino acids 27 and 34 in exon 2 (2–4). Thus, we analyzed a part of the exon 2 in H3F3A gene by polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) analysis. Genomic DNA each from tumor cells and normal cells of the same patients were amplified by PCR with a primer pair (5′CAC CCAGGAAGCAACTG -3′ and 5′TTCCTGTTATCCAT CTTTTTGT-3′; product size: 164 base pairs). Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by autoradiogram. Other procedures of the PCRSSCP and DNA sequencing were described in our previous studies (8). SSCP analysis of aberrantly migrating bands on the SSCP led to the identification of one H3F3A somatic mutation in a childhood precursor B-ALL, but no H3F3A mutation was detected in the other tumors. However, the H3F3A mutation detected was a mutation in the intron (c.128 + 30_31delAA) that would not alter the coding sequences. To confirm the SSCP results, we repeated our experiments twice, including PCR and SSCP and to ensure the specificity of the results, and found that the data were consistent. A main concern in cancer research is to identify whether any mutation found in a cancer type is common in other cancer types as well. Because H3F3A is involved in chromatin remodeling that is essential not only for glial cells but also for hematopoietic cells, we analyze H3F3A somatic mutation in hematologic neoplasia. However, the present study detected somatic mutation of H3F3A only in a BALL, but its incidence was very low (0.5% in the childhood ALL). Moreover, this mutation was an intron mutation and not overlapped with the known H3F3A mutations. Taken together, our results suggest that the H3F3A codon 27 and 34 mutations may be a very rare event in human hematopoietic tumors. The discovery of the H3F3A mutation offered Table 1 H3F3A exon 2 mutation analyzed in hematopoietic tumors

STAT3 exon 21 mutation is rare in common human cancers.

Abstract: To the EditorTo find cancer-related genome alterations in T-cell large granular lymphocytic leukemia, Koskela et al. [1] analyzed whole exomes of the cancer cells by next-generation exome sequencin...

Mutation analysis of DICER1 gene in hematologic tumors.

Abstract: MicroRNAs (miRNAs) regulate gene expression by pairing to mRNA of the target genes [1]. Dicer, an endonuclease (RNase III), is a protein involved in miRNA biogenesis [1]. During biogenesis, precursor miRNAs are processed by Dicer to generate a short RNA duplex [1]. Mounting evidence indicates that not only miRNAs themselves, but also the genes involved in miRNA biogenesis, play important roles in cancer pathogenesis [1]. Heravi-Moussavi et al . [2] recently identifi ed frequent somatic mutations of DICER1 , the gene encoding Dicer protein, in Sertoli – Leydig cell tumors (60%), and less frequent mutations in juvenile granulosa cell tumors, yolk sac tumors, mature teratomas, testicular non-seminomatous germ cell tumors and embryonal rhabdomyosarcomas. Th e mutations were recurrent in exons 25 and 26 of DICER1 , and most of the mutations were missense mutations occurring at Asp 1709 and Glu 1813 residues [2]. Because miRNA biogenesis and regulation are ubiquitous to all cell types, it is important to know whether any other types of human tumors carry DICER1 recurrent mutations. However, to date, the mutation status of the DICER1 gene in hematologic tumors remains unknown. In the present study, we attempted to determine whether the DICER1 gene is somatically mutated in acute leukemias and other common hematologic tumors. For this, we analyzed somatic mutations of DICER1 gene in fresh bone marrow aspirates of 798 hematologic tumors (acute myelogenous leukemia [AML], acute lymphoblastic leukemia [ALL], multiple myeloma and myelodysplastic syndrome) (Table I) by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) assay. Also, we analyzed the gene in paraffi n-embedded tissues of 132 non-Hodgkin lymphomas (NHLs). Approval was obtained from Th e Catholic University of Korea, College of Medicine ’ s institutional review board for this study. To date, all of the recurrent DICER1 somatic mutations in Sertoli – Leydig cell tumors have been detected in exons 25 and 26 [2]. Genomic DNA each from tumor cells and normal cells of the same patients were amplifi ed by PCR with specifi c primer pairs for exon 25 (forward and reverse, respectively: 5 ’ -ATGTGGGGATAGTGTAAATGC-3 ’ and 5 ’ -GGGTCTTCATAAAGGTGCTT-3 ’ ) and exon 26 (5 ’ -GGCCTTTTTGCTTACAAGTCA-3 ’ and 5 ’ -GGGATAGTACACCTGCCAGAC-3 ’ ). Radioisotope ([ 32 P]dCTP) was incorporated into the PCR products for detection by autoradiography. Other procedures of the PCR-SSCP and DNA sequencing were described in our previous studies [3]. On the SSCP autoradiogram, all of the PCR products were clearly seen. SSCP analysis of aberrantly migrating bands led to the identifi cation of one DICER1 somatic mutation in an NHL (a diff use large B-cell lymphoma), but no DICER1 mutation was detected in the other tumors. Th e mutation was a missense mutation that substituted Glu 1813 residue (c.5438A C [p.Glu1813Ala]) (Figure 1). Importantly, this mutation was the same mutation as previously reported in Sertoli – Leydig cell tumors [2]. We repeated the experiments twice, including PCR-SSCP and direct DNA sequencing analysis to ensure the results, and found that the data were consistent. A main concern in cancer research is to identify whether any mutation found in a cancer type is common in other cancer types as well. In the present study, we analyzed DICER1 somatic mutation in various hematologic tumors. However, the prevalence of the DICER1 mutation in exons 25 and 26 was very low (0.1% of samples). Taken together, our results suggest that DICER1 exons 25 and 26 mutations may be very

Abstract 1921: Mutational and expressional analyses ofDICER1gene in ovarian stromal tumors and other common tumors.

Abstract: Recurrent missense somatic mutations in DICER1 gene affecting exons 25 and 26 have recently been reported in non-epithelial tumors of ovary, especially in Sertoli-Leydig cell tumors (SLCT). Such a recurrent nature of the mutations strongly suggests that DICER1 mutation may play an important role in the development of ovarian non-epithelial tumors. The aim of this study was to further characterize the DICER1 mutations in human tumor tissues. We analyzed the DICER1 mutations in 2,819 tumor tissues from ovary and other organs by single-strand conformation polymorphism analysis. We found the DICER1 exon 25 and 26 hot-spot mutations in 2 of 14 SLCT (14.3%) and one of 132 non-Hodgkin lymphomas (0.8%), but none in other tumors. In SLCT, DICER1 expression was observed in Sertoli cells, but neither in Leydig nor spindle-shaped cells. We also analyzed DICER1 expression in lung, stomach, colon and prostate cancer tissues. DICER1 expression was lost in 37% of prostate cancers, but not in lung, stomach and colon cancers. Our data indicate that DICER1 recurrent mutations commonly occur in SLCT, but the incidence was significantly lowerthan that in the earlier study. Our data suggest that both DICER1 mutation and its expression loss may be important in tumor pathogenesis and that they are tumor type-specific. Citation Format: Min Sung Kim, Eun Mi Je, Youn Jin Choi, Nam Jin Yoo, Sug Hyung Lee. Mutational and expressional analyses of DICER1 gene in ovarian stromal tumors and other common tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1921. doi:10.1158/1538-7445.AM2013-1921

Alteration of p62/SQSTM1 Expression Is Uncommon in Gastrointestinal and Prostate Cancer Tissues

Abstract: Sequestosome 1 (p62/SQSTM1) contains multiple domains that interact with key components in cellular processes and signaling, including autophagy, nuclear factor-κB (NF-κB) activation, and oxidative stress signaling.1 p62 behaves as an adaptor between autophagic machinery and ubiquitinated proteins and promotes autophagic degradation of ubiquitinated targets. Altered autophagy is accompanied by p62 accumulation and ubiquitinated proteins.1 p62 activates NF-κB signaling through binding with TRAF6 and RIP1. Additionally, by interacting with the Nrf2 binding site in Keap1, p62 competitively inhibits the Nrf2-Keap1 interaction, thereby activating the transcription of Nrf2 target genes that induces antioxidant responses.1 With respect to disease involvement, the p62-encoding gene is frequently mutated in Paget's disease of bone.1,2 With respect to cancer, p62 is involved in many signaling pathways whose functions are important in cancer development. p62 acts either as a tumor suppressor (via the Wnt pathway) or a pro-oncogenic protein (via the NF-κB and mammalian target of rapamycin pathways).1,2 p62 is highly expressed in breast, lung, gastric, colon, esophageal, and hepatocellular carcinomas and is associated with poor cancer patient prognosis.3-6 In contrast, a recent study reported that p62 was downregulated in colon carcinomas.7 The aim of our study was to confirm whether p62 expression is altered in gastric and colorectal cancers and to address whether it is altered in other cancers. To determine whether alterations of p62 expression are present in other human cancers, we analyzed the expression of p62 in gastric cancer (GC), colorectal cancer (CRC), and prostate cancers (PCa) by immunohistochemistry using 11 tissue microarray (TMA) blocks. Cases of CRC (n=103) originated from the cecum (n=2), ascending colon (n=19), transverse colon (n=6), descending colon (n=4), sigmoid colon (n=28), and rectum (n=44). Cases of GC (n=100) consisted of 50 diffuse, 36 intestinal, and 14 mixed-type GC by Lauren classification, as well as four early GC and 96 advanced GC cases according to the depth of invasion. The PCa cases (n=107) consisted of one case with a Gleason score of 5, 10 with a score of 6, 47 with a score of 7, 10 with a score of 8, and 39 with a score of 9. For immunohistochemistry, we used the ImmPRESS System (Vector Laboratories, Burlingame, CA, USA) with a rabbit polyclonal antibody against human sequestosome 1 (Thermo Scientific, Rockford, IL, USA; dilution 1/2,000). By visual inspection under a microscope, we graded the immunoreactivity as -, +, or ++. Tumor and normal tissues were interpreted as positive by immunohistochemistry when they scored either + or ++. Other procedures for mutation and immunohistochemistry have been described in our previous reports.8,9 In the immunohistochemistry analysis, normal gastric mucosal epithelial, colonic mucosal epithelial, and prostate glandular cells exhibited positive p62 immunostaining in all cases (Fig. 1). All of the cases exhibited ++ immunopositivity. In the tumors, immunopositivity for p62 was observed in 97 cases (+, three; ++, 94 cases) of GC, 97 cases (+, two; ++, 95 cases) of CRC, and 100 cases (+, two; ++, 98 cases) of PCa. There was no significant difference in the immunopositivity between normal and GC, CRC, and PCa tumor tissues (Fisher exact test, p>0.05). There was no significant difference in the immunopositivity between the GC, CRC, and PCa tissues (Fisher exact test, p>0.05). There was no association between p62 expression and clinicopathological parameters, including Gleason score, invasion/metastasis, and TNM stage (chi-square test, p>0.05). Because p62 is involved in diverse cancer-related signaling, p62 expression has been studied in many cancers. However, our results demonstrated that p62 was expressed well in both normal and tumor cells of GC and CRC tissues and that there was no difference in the expression with respect to the clinicopathological data. Our data from Korean patients are in agreement with earlier data that demonstrated that both normal and cancer cells in gastric and colon cancers from Chinese patients expressed p62.6 However, these data are different from other data obtained in a Canadian population that demonstrated decreased expression of p62 in colon cancers compared with the high expression of p62 in normal colonic epithelial cells.7 One possibility for the disagreement may be ethnic differences (Asian vs Caucasian). Another explanation may be the method we used. Many researchers are concerned about the potential bias in TMAs, mostly due to the heterogeneity of expression patterns in TMA. One way to reduce bias in TMAs is to confirm the data in conventional large tissue sections. To validate the TMA data, we further analyzed the expression in another series of 20 additional colorectal adenocarcinomas and their normal mucosa tissue sections that were more than 1 cm2 in size and found that p62 was similarly expressed in the tumors (95%) and in the normal mucosa tissues (positive immunostaining in all cases). We also found that p62 expression was not altered in PCa. Our data indicate that alteration of p62 expression is not a common feature in all cancers and that p62 alterations may be tissue-specific. Fig. 1 Visualization of p62 expression in gastric, colorectal, and prostate cancer tissues by immunohistochemistry. (A) Normal gastric epithelial cells exhibit positive immunostaining. (B) In a case of gastric cancer, the cancer cells exhibit p62 immunostaining. ...

Frameshift mutation of a tumor suppressor gene PALB2 in gastric and colorectal cancers with microsatellite instability.

Abstract: To the Editor Partner and localizer of BRCA2 (PALB2), also known as FANCN, binds to BRCA2 and contributes to DNA homologous recombination repair and cancer suppression (1). PALB2 is considered a candidate tumor suppressor gene and is inactivated in some cancers. Germline mutations of PALB2 gene are associated with familial breast and familial pancreatic cancers (2). Also, promoter hypermethylation of the PALB2 gene occurs in both sporadic and familial breast/ovarian cancers (3). However, it remains unknown whether PALB2 is inactivated in other cancers besides breast and pancreatic cancers. By analyzing human gene sequences in a public database (http://genome.cse.ucsc.edu/), we found that PALB2 has a mononucleotide repeat in the coding sequences (A7 repeat in exon 4) that could be a target for frameshift mutation in the cancers with microsatellite instability (MSI). It is well known that 10–30% of in gastric (GC) and colorectal (CRC) cancers with defective DNA mismatch repair (MMR) show microsatellite instability (MSI). In this study, we analyzed their mononucleotide repeats in 46 GC and 55 CRC by polymerase chain reaction (PCR) and singlestrand conformation polymorphism (SSCP) assay as described previously (4, 5). The GC consisted of 32 GC with high MSI (MSI-H), 14 GC with low MSI (MSI-L), 40 CRC with MSI-H, and 15 CRC with MSI-L. Genomic DNA each from tumor cells and corresponding normal cells were amplified with a specific primer pair by PCR. Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by SSCP autoradiogram. On SSCP, we observed aberrant bands in four cancers in the PALB2 gene (Fig 1). DNA from their corresponding normal tissues showed no migration shifts in the SSCP, indicating that the aberrant bands had risen somatically. DNA sequencing analysis confirmed that aberrant bands represented a somatic mutation. All of the mutations were a recurrent frameshift mutation that showed deletion of an ‘A’ in the ‘A7’ repeat (c.886delA). This mutation would result in a truncation of the PALB2 protein (p.Met296X). The mutation was detected in three GC with MSI-H (3/32; 9.4%) and one CRC with MSI-H (1/40; 2.5%), but none in those with MSI-L. There was no significant difference of the mutations with respect to stage or histologic types of the cancers (Fisher’s exact test, p>0.05). Most of the PALB2 mutations identified in germline breast or pancreatic cancer are frameshift mutations that truncate the full-length PALB2 protein (2). Similarly, the truncating mutation identified in this study would lead to premature stops of amino acid syntheses in PALB2 protein and hence resembles a typical loss-of-function mutation. Although we did not investigate functional implications of the PALB2 mutation in cancer, it may inactivate the tumor suppressor functions of PALB2. Our report here is the first report that shows

Loss of RNA-induced silencing complex-related proteins in non-small cell lung cancers.

Abstract: To the Editor: MicroRNAs (miRNAs), a class of endogenous non-protein-coding RNA molecules, regulate gene expression by pairing with messenger RNA of target genes (1). Mounting evidence indicates that both miRNAs and miRNA biogenesis-related proteins are frequently deregulated in cancers. For example, recurrent mutations of DICER1 gene have been detected in Sertoli-Leydig cell tumor of ovary (2). Ago proteins and TNRC proteins are main components of RNA-induced silencing complex (RISC) in the miRNA biogenesis (3, 4). Both Ago2 and TNRC6A genes are mutated in gastric and colorectal cancers with microsatellite instability (5). Also, loss of Ago2 and TNRC6A proteins was observed in gastric and colorectal cancers (5). In contrast, increased expression of Ago2 and TNRC6A proteins was identified in prostate and esophageal cancers (6). To extend the knowledge on miRNA biogenesis-related proteins in cancers, we analyzed Ago2 and TNRC6A protein expression in non-small cell lung cancers (NSCLCs) in this study. For this, we used formalin-fixed tissues of 45 NSCLCs {23 adenocarcinomas [AD] and 22 squamous cell carcinomas (SCC)}. Ages of the NSCLC patients ranged 36–79 years with an average of 57 years. For immunohistochemistry, we used ImmPRESS Polymer Detection System (Vector, Berlingame, CA, USA). Antibodies for human Ago2 (Abcam, Cambridge, UK; dilution 1/100) and human TNRC6A (Sigma, Saint Louis, MO, USA; dilution 1/50) were used as primary antibodies. Sections were incubated overnight at 4 °C with each antibody. The immunohistochemistry was performed as described previously (5, 6). Tumors were interpreted as positive by immunohistochemistry when at least weak to intense staining was seen in the cells. The results were reviewed independently by two pathologists. As negative controls, a slide was treated by replacement of primary antibody with a blocking reagent. The immunostaining was judged to be specific by absence of consistent immunostaining of cells by replacement of primary antibody with the blocking reagent. In normal bronchi and bronchioles, the epithelial cells were positive for both Ago2 and TNRC6A immunostaining (Figs 1A and B). In NSCLCs, expressions for Ago2 and TNRC6A were found in 30 (67%) and 36 (80%) cancers, respectively (Figs 1C–F). According to the histological subtypes, there was no difference of Ago2 or TNRC6A expression between AD and SCC (Fisher’s exact test, p > 0.05) (Table 1). The Ago2 and TNRC6A immunostainings were identified in cytoplasm and nuclei/cytoplasm in the cells, respectively (Fig 1). The immunostainings of Ago2 and TNRC6A were not different with respect to location in the tissue sections. Next, we analyzed relationship of each immunostaining with various pathologic parameters (age, sex, tumor size and stage). However, there was no significant association (v test, p > 0.05). Although several studies identified alterations of components in the miRNA biogenesis in various cancers, their status in NSCLC remains largely unknown. In this study, we sought to determine whether there was any difference of Ago2 and TNRC6A expressions between normal and cancer cells in lung tissues, which had not been studied. Whereas both Ago2 and TNRC6A were well expressed in the normal bronchial epithelial cells, they were not expressed in some fractions of NSCLC (Table 1). These data suggests that loss of Ago2 and TNRC6A expression may be a feature of NSCLC. Our earlier studies and this study have found that Ago2 and TNRC6A expressions were different with respect to cancer types (5, 6), suggesting a possibility that miRNA biogenesis in cancers might be quite variable in cancers. To date, many of miRNA data have been analyzed in NSCLC (7). Based on the alterations and functions in miRNA regulation, it is necessary to further analyze the miRNA data in NSCLC together with miRNA

a Regulator of Procaspase-1 Activation Is Containing a Caspase Recruitment Domain Apoptosis-Associated Speck-Like Protein

Abstract: Angela Stassinopoulos, Junji Sagara and John C. ReedChristian Stehlik, Sug Hyung Lee, Andrea Dorfleutner,http://www.jimmunol.org/content/171/11/6154J Immunol€2003; 171:6154-6163; ;Referenceshttp://www.jimmunol.org/content/171/11/6154.full#ref-list-1This article cites 41 articles, 20 of which you can access for free at: Subscriptionshttp://jimmunol.org/subscriptionsInformation about subscribing to The Journal of Immunology is online at: Permissionshttp://www.aai.org/ji/copyright.htmlSubmit copyright permission requests at: Email Alertshttp://jimmunol.org/cgi/alerts/etocReceive free email-alerts when new articles cite this article. Sign up at: