Chang Hyeok Ahn

Frameshift mutations of autophagy-related genes ATG2B, ATG5, ATG9B and ATG12 in gastric and colorectal cancers with microsatellite instability.

Mounting evidence indicates that alterations of autophagy processes are directly involved in the development of many human diseases, including cancers. Autophagy‐related gene (ATG) products are main players in the autophagy process. In humans there are 16 known ATG genes, of which four (ATG2B, ATG5, ATG9B and ATG12) have mononucleotide repeats with seven or more nucleotides. Frameshift mutations of genes with mononucleotide repeats are features of cancers with microsatellite instability (MSI). It is not known whether ATG genes with mononucleotide repeats are altered by frameshift mutations in gastric and colorectal carcinomas with MSI. For this, we analysed the mononecleotide repeats in ATG2B, ATG5, ATG9B and ATG12 in 32 gastric carcinomas with high MSI (MSI‐H), 13 gastric carcinomas with low MSI (MSI‐L), 43 colorectal carcinomas with MSI‐H and 15 colorectal carcinomas with MSI‐L by a single‐strand conformation polymorphism (SSCP) analysis. We found ATG2B, ATG5, ATG9B and ATG12 mutations in 10, 2, 13 and 0 cancers, respectively. The mutations were detected in MSI‐H cancers but not in MSI‐L cancers. Gastric and colorectal cancers with MSI‐H harboured one or more ATG mutations in 28.1% and 27.9%, respectively. Our data indicate that frameshift mutations in ATG genes with mononucleotide repeats are common in gastric and colorectal carcinomas with MSI‐H, and suggest that these mutations may contribute to cancer development by deregulating the autophagy process. Copyright

Expression of beclin‐1, an autophagy‐related protein, in gastric and colorectal cancers

Autophagy plays important roles in both cell death and cell survival. Beclin‐1, a key regulator of autophagy formation, has been considered as a haploinsufficient tumor suppressor. Loss of expression or point mutation could serve as a mechanism of loss of beclin‐1 tumor suppressor function in cancers. However, our recent study revealed that point mutation of the beclin‐1 gene is a rare event in common human cancers. In this study we investigated beclin‐1 protein expression in 103 colorectal and 60 gastric carcinoma tissues by immunohistochemistry using a tissue microarray approach. In the cancers, expression of beclin‐1 was detected in 95% of the colorectal carcinomas and 83% of the gastric carcinomas. In contrast, normal mucosal cells of both stomach and colon showed no or very weak expression of beclin‐1. There was no significant association of beclin‐1 expression with clinocopathologic characteristics, including invasion, metastasis and stage. The beclin‐1 expression of colorectal and gastric cancers in the present study is quite in contrast to that of the breast cancers in the previous study, which showed a decreased beclin‐1 expression in breast cancer cells compared to normal breast cells. Our data indicate that beclin‐1 inactivation by loss of expression may not occur in colorectal and gastric cancers. Rather, increased expression of beclin‐1 in the malignant colorectal and gastric epithelial cells compared to their normal mucosal epithelial cells suggests that neo‐expression of beclin‐1 may play a role in both colorectal and gastric tumorigenesis.

Frameshift mutation of UVRAG, an autophagy-related gene, in gastric carcinomas with microsatellite instability☆

Alteration of autophagy is involved in tumor development. Beclin1, an important regulator of autophagy, acts as a tumor suppressor. Ultraviolet (UV) radiation resistance-associated gene (UVRAG) binds with Beclin1 and induces autophagy. There is a polyadenine tract in UVRAG gene (A10 in exon 8) that is a target for frameshift mutations in colorectal carcinomas with microsatellite instability (MSI). Functionally, colon cancer cells with the frameshift mutation of UVRAG show reduced autophagy formation and increased tumorigenicity. The aim of this study was to determine whether the frameshift mutations of UVRAG are also present in gastric carcinomas with MSI. For this, we analyzed human UVRAG exon 8 in 45 gastric carcinomas with MSI and 92 gastric carcinomas without MSI by a single-strand conformation polymorphism analysis. Overall, we detected 3 frameshift mutations of UVRAG in the polyadenine tract (3/45; 6.7%), and all of them were found in MSH-high (H) subtypes (3/32; 9.4%). The 3 mutations consisted of 2 c.708_709delA and 1 c.709delA which would result in premature stops of the UVRAG protein synthesis. The present data indicate that frameshift mutations in the polyadenine tract in UVRAG gene are present in gastric carcinomas as well and suggest that the affected gastric cancer cells with the mutations may have a reduced autophagy activity.

Mutational analysis of caspase 1, 4, and 5 genes in common human cancers

Mounting evidence indicates that deregulation of apoptosis is involved in the mechanisms of cancer development. Mutations of genes encoding caspases, the executioners of apoptosis, have been detected in human cancers, indicating inactivation of apoptosis by the mutations of caspase is an important mechanism in cancer development. The aim of this study was to see whether genes encoding human caspases 1, 4, and 5 are mutated in human cancers. We analyzed the entire coding region and all splice sites of human caspase 1, 4, and 5 genes for the detection of somatic mutations in 337 human cancers, including 103 colorectal, 54 gastric, 60 breast, 60 hepatocellular, and 60 lung carcinomas by a single-strand conformation polymorphism assay. We detected 2 (0.6%) caspase-1, 2 (0.6%) caspase-4, and 15 (4.4%) caspase-5 mutations in the 343 cancers. The mutations were detected in 11 gastric carcinomas (2 caspase-1 and 9 caspase-5 mutations), 6 colorectal carcinomas (2 caspase-4 and 4 caspase-5 mutations), 1 breast carcinoma (1 caspase-5 mutation), and 1 lung carcinoma (1 caspase-5 mutation). The mutations consisted of 11 mutations in exons and 8 mutations in noncoding sequences. The 11 mutations in the exons consisted of 3 missense, 1 silent, and 7 frameshift mutation(s). Of note, most (6/9) of the caspase-5 mutations in the coding sequences were detected in microsatellite instability (MSI)-positive cancers. These data indicate that somatic mutations of caspase-1 and caspase-4 genes are rare in common solid cancers. In addition, the data indicate that caspase-5 gene is commonly mutated in the MSI-positive cancers, and suggest that inactivation of caspase-5 may play a role in the tumorigenesis of MSI-positive cancers.

Mutational Analysis of TTK Gene in Gastric and Colorectal Cancers with Microsatellite Instability

PURPOSE The TTK gene plays a crucial role in regulation of the mitotic checkpoint. The TTK gene has an A9 mononucleotide repeat in the coding sequences, which harbors mutations in gastric (GC) and colorectal cancers (CRC) with microsatellite instability (MSI). However, there are three more repeats (the A7s) in the coding sequences that have not been analyzed. The aim of this study was to explore whether the three A7s as well as the A9 are altered in GC and CRC, and to find any association of TTK mutation with clinocopathologic characteristics of GC and CRC. MATERIALS AND METHODS We analyzed exon 5 (A7 and A7) and exon 22 (A9 and A7) which have repeat sequences in 30 GC with high MSI (MSI-H), 15 GC with low MSI (MSI-L), 35 CRC with MSI-H, and 15 CRC with MSI-L, by single-strand conformation polymorphism (SSCP) and DNA sequencing assays. RESULTS Overall, we detected 23 frameshift mutations in the repeat sequences of TTK in the GC with MSI-H (11/30; 36.7%) and the CRC with MSI-H (12/35; 34.3%), but not in the cancers with MSI-L. The mutations were observed in both A9 and A7 of exon 22, but in neither of the two A7s of exon 5. The mutations consisted of c.2560delA, c.2560dupA, c.2571delA and c.[2560delA(+)2571delA]. All of the mutations were frameshift mutations and would result in premature stops of TTK protein synthesis. There was no significant difference in clinopathologic parameters of the cancers with the mutations. CONCLUSION Our data indicate that frameshift mutations of TTK are common in both GC and CRC with MSI-H, and that the mutations occur not only in the A9 repeat but also in the A7 repeat. The data suggest that frameshift mutations of TTK might alter cell cycle control in the affected cells and contribute to pathogenesis of cancers with MSI-H.

Mutational analysis of CASP10 gene in colon, breast, lung and hepatocellular carcinomas

Aims: Evasion of apoptosis is a feature of cancer cells. As a mechanism of apoptosis inactivation in cancer cells, somatic mutations of pro‐apoptotic genes have been reported in many cancers. Caspase‐10 is an initiation‐phase caspase, and somatic mutation of CASP10 that encodes caspase‐10 has been found in non‐Hodgkins lymphoma and gastric carcinoma. Methods: The aim of this study was to explore whether CASP10 gene is somatically mutated in colon, breast, lung, and hepatocellular carcinomas. We analysed the entire coding region and all splice sites of CASP10 in 47 colon, 47 breast, 47 lung, and 47 hepatocellular carcinomas by a single‐strand conformation polymorphism (SSCP) assay. Results: We found two CASP10 mutations in the colon cancers (2/47; 4.3%), but none in breast, lung or hepatocellular carcinomas. One mutation [c.41A > C (p.Lys14Thr)] was a missense mutation, while the other was a substitution mutation in a splice site (c.684 + 4G > A). The colon cancer with the CASP10 missense mutation harboured additional CASP gene mutations (CASP3, 7 and 8). Conclusion: Our data indicate that somatic mutation of CASP10 is rare in colon, breast, lung, and hepatocellular carcinomas. However, the data also suggest that CASP10 mutation might contribute to the pathogenesis of some colon carcinomas together with other CASP gene mutations.

Absence of MLL3 mutations in colorectal carcinomas of Korean patients

A central aim of cancer research has been to identify mutated genes that are causally implicated in tumorigenesis. Mutation in cancer could be categorized either as cancer-specific mutation affecting a key gene in the neoplastic process or as non-functional passenger mutation (1). Analyses of human genes to find functional cancer-specific mutations have focused on a small fraction of genes or domains. However, recently Sjöblom et al. analyzed 13,023 genes (approximately one third of the total number of human genes), a set of protein coding genes termed the consensus coding sequences (2). In 35 breast and 35 colorectal cancer tissues, they identified 189 genes that were mutated at significant frequencies. In addition to genes that had been reported to be mutated in colorectal or breast cancers, such as TP53, APC and KRAS, many other genes were newly found to be mutated in these cancers (2). Of these, myeloid/lymphoid or mixed-lineage leukemia 3 (MLL3) was found to be frequently mutated in colorectal carcinomas (6/35; 17.1%). MLL3 has a significant homology with MLL that is frequently fused with other gene products by chromosomal translocations (3). The six mutations identified in the MLL3 gene consisted of five substitution mutations (four missense mutations and one nonsense mutation) and one deletion mutation. The aim of this study was to confirm the presence of MLL3 gene mutations in colorectal carcinomas, and to see whether there exists any significant difference in the frequency of MLL3 mutations between colorectal carcinomas in the USA (2) and Korea. Methacarn-fixed tissues of 104 colorectal carcinomas from Koreans were

Immunohistochemical analysis of phospho‐BAD protein and mutational analysis of BAD gene in gastric carcinomas

Mounting evidence indicates that deregulation of apoptosis contributes to the development of human cancers. BAD, a proapoptotic Bcl‐2 family protein, regulates the intrinsic apoptosis pathway. The aim of this study was to explore whether alterations of phospho‐BAD (p‐BAD) protein that antagonizes apoptosis function of BAD and mutation of BAD gene are characteristics of human gastric cancers. We analyzed expression of p‐BAD in 60 gastric adenocarcinomas by immunohistochemistry. Also, we analyzed BAD gene for detection of somatic mutations by single‐strand conformation polymorphism (SSCP) assay. p‐BAD expression was detected well in normal gastric mucosal epithelial cells, whereas it was detected in only 51% (31 of the 60) of the cancers. There was no somatic mutation of BAD gene in the 60 gastric cancer samples. The decreased expression of p‐BAD in malignant gastric epithelial cells compared to normal mucosal epithelial cells suggested that loss of p‐BAD expression may play a role in gastric tumorigenesis. The data also suggest that BAD mutation may not be a direct target of inactivation in gastric tumorigenesis.

Somatic mutation of PIK3R1 gene is rare in common human cancers

1Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Korea, 2Hematology-Oncology Clinic, National Cancer Center of Korea, Goyang, Korea, 3Center for Lung Cancer, National Cancer Center of Korea, Goyang, Korea, 4Department of General Surgery, College of Medicine, The Catholic University of Korea, Seoul, Korea and 5Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea

Increased expression of endonuclease G in gastric and colorectal carcinomas.

Aims Endonuclease G (EndoG) is a mitochondrial protein that plays a role in DNA fragmentation during apoptosis. In addition, EndoG plays a role in cell proliferation and survival. It may be important to identify EndoG protein expression to predict its function in human cancers. The aim of this study was to explore whether alteration of EndoG expression might be a characteristic of colorectal or gastric carcinoma. Methods We investigated EndoG protein expression in 103 colorectal and 60 gastric carcinoma tissues by immunohistochemistry using a tissue microarray approach. Results Expression of EndoG was detected in 72 (70%) of the colorectal carcinomas and 41 (68%) of the gastric carcinomas in cytoplasm. By contrast, normal mucosal cells of both stomach and colon tissues showed no or very weak expression of EndoG. There was no significant association of EndoG expression with clinocopathological characteristics, including invasion, metastasis and stage. Conclusion Our data indicate that EndoG inactivation by loss of expression may not occur in colorectal and gastric cancers. Rather, increased expression of EndoG in colorectal and gastric cancer cells compared to their normal mucosal epithelial counterparts suggests that neo-expression of EndoG may play a role in both colorectal and gastric tumorigenesis.