Eun Goo Jeong

Somatic Mutations of JAK1 and JAK3 in Acute Leukemias and Solid Cancers

Purpose: The aim of this study was to see whether JAK1, JAK3, and TYK2 genes are altered in human cancers. Experimental Design: We analyzed 494 tissues from 186 acute adulthood leukemias, 30 multiple myelomas, and 278 common solid cancers, including 90 breast, 47 gastric, 47 colon, 47 lung, and 47 hepatocellular carcinomas by single-strand conformation polymorphism analysis. Results: Overall, we found six JAK1 mutations (four in acute leukemias, one in a lung carcinoma, and one in a breast carcinoma) and three JAK3 mutations (two in breast carcinomas and one in a gastric carcinoma). Of note, three JAK1 mutations were an identical p.V658F mutation, which is homologous to JAK2 p.V617F mutation. We also found two other JAK1 mutations that occurred at very close sites (p.T782M and p.L783F). We found three of the four leukemias with JAK1 mutations expressed mutated JAK1 at the mRNA level. For JAK3 mutations, one of them was JAK3 p.V715I that is homologous to the JAK1 p.L783F. These recurrent mutations in identical and homologous sites suggest a possibility that alterations of these amino acids might be important for tumor pathogenesis. With respect to the cancer types, T-acute lymphoblastic leukemia (T-ALL) showed the highest incidence of the mutations (3 of 11; 27.3%). Conclusion: Our data indicate that both JAK1 and JAK3 mutations occur in common human cancers and that JAK1 mutation in T-ALL is a frequent event. The data suggest that some of the JAK1 and JAK3 mutations may to be functional and contributes to cancer development, especially to T-ALL development.

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 NOTCH1, 2, 3 and 4 genes in common solid cancers and acute leukemias

NOTCH proteins (NOTCH1, NOTCH2, NOTCH3 and NOTCH4) play crucial roles in embryonic development. Also, mounting evidence indicates that NOTCH contributes to the pathogenesis of hematopoietic and solid malignancies. Recent studies reported a high incidence of gain‐of‐function mutations of the NOTCH1 gene in T‐cell acute lymphoblastic leukemias (ALL). To see whether NOTCH1 mutation occurs in other malignancies, we analyzed NOTCH1 for the detection of somatic mutations in 334 malignancies, including 48 lung, 48 breast, 48 colorectal and 48 gastric carcinomas, and 142 acute leukemias (105 acute myelogenous leukemias, 32 B‐ALLs and 4 T‐ALLs) by single‐strand conformation polymorphism assay. Also, to see whether other NOTCH genes harbor somatic mutations, we analyzed NOTCH2, NOTCH3 and NOTCH4 genes in the same tissue samples. Overall, we detected three NOTCH mutations in the cancers, which consisted of one NOTCH1 mutation in the T‐ALLs (25.0%), one NOTCH2 mutation in the breast carcinomas (2.1%), and one NOTCH3 mutation in the colorectal carcinomas (2.0%). There was no NOTCH mutation in other malignancies analyzed. Our data indicate that NOTCH1 is mutated in T‐ALL, but not in other common human cancers, and that NOTCH2, NOTCH3 and NOTH4 genes are rarely mutated in common human cancers. Despite the importance of NOTCH activation in many types of human cancers, mutation of NOTCH genes, except for NOTCH1 mutation in T‐ALL, may not play an important role in the tumorigenesis of common cancers.

Decreased expression of tumour suppressor Bax-interacting factor-1 (Bif-1), a Bax activator, in gastric carcinomas

Aims: Evasion of apoptosis is one of the hallmarks of cancer. Bax‐interacting factor‐1 (Bif‐1) interacts with both Bax and Bak that are essential for the intrinsic pathway of apoptosis, and enhances apoptosis. The aim of this study was to explore whether alteration of Bif‐1 expression might be a characteristic of gastric cancer. Methods: We analysed the expression of Bif‐1 protein in 60 gastric adenocarcinomas by immunohistochemistry using a tissue microarray approach. Results: In normal gastric mucosal cells, surface and glandular epithelial cells strongly expressed Bif‐1. While Bif‐1 expression was detected in 24 cases (40%) of the gastric carcinomas, there was no Bif‐1 immunostaining in the remaining 36 cancers. Even in the 24 cases with positive Bif‐1 immunostainings, 10 cancers showed decreased intensity of immunostaining compared with the normal mucosal epithelial cells. Conclusion: The decreased expression of Bif‐1 in malignant gastric epithelial cells compared with the normal mucosal cells suggests that loss of Bif‐1 expression may play a role in gastric tumorigenesis, possibly by inhibiting the apoptosis mediated by Bif‐1.

Detection of Low-Level KRAS Mutations Using PNA-Mediated Asymmetric PCR Clamping and Melting Curve Analysis with Unlabeled Probes

Detection of somatic mutations in clinical cancer specimens is often hampered by excess wild-type DNA. The aim of this study was to develop a simple and economical protocol without using fluorescent probes to detect low-level mutations. In this study, we combined peptide nucleic acid (PNA)-clamping PCR with asymmetric primers and a melting curve analysis using an unlabeled detection probe. PNA-clamping PCR, which suppressed amplification of the wild-type allele, was more sensitive for KRAS codon 12 mutation detection than nonclamping PCR in 5 different mutant cell lines. Three detection probes were tested (a perfectly matched antisense, a mismatched antisense, and a mismatched sense), and the mismatched sense detection probe showed the highest sensitivity (0.1% mutant detection) under clamping conditions. With this probe, we were able to detect not only the perfectly matched KRAS mutation, but also 4 other mismatched mutations of KRAS. We then applied this protocol to 10 human colon cancer tissues with KRAS codon 12 mutations, successfully detecting the mutations in all of them. Our data indicate that the combination of perfectly matched antisense PNA and a mismatched sense detection probe can detect KRAS mutations with a high sensitivity in both cell lines and human tissues. Moreover, this study might prove an easily applicable protocol for the detection of low-level mutations in other cancer genes.

Expression of NEDD-1, a PTEN regulator, in gastric and colorectal carcinomas

Recent studies have disclosed that NEDD4‐1 regulates PTEN activity by ubiquitination. NEDD4‐1 negatively regulates PTEN in cytosol and acts as an oncogenic protein. By contrast, NEDD4‐1 promotes PTEN nuclear import and acts as a tumor suppressor. Despite the importance of NEDD4‐1 in PTEN regulation in cancer cells, expression of NEDD4‐1 protein in cancer tissues is unknown. The aim of this study was to analyze NEDD4‐1 expression in colorectal and gastric cancer tissues. We investigated NEDD4‐1 protein expression in 103 colorectal and 60 gastric carcinoma tissues by immunohistochemistry using a tissue microarray approach. In the cancers, expression of NEDD4‐1 was detected in 82 (80%) of the colorectal carcinomas and 45 (75%) of the gastric carcinomas in cytoplasm. By contrast, the normal mucosal cells of both stomach and colon showed no or very weak expression of NEDD4‐1. There was no significant association of NEDD4‐1 expression with clinicopathologic characteristics, including invasion, metastasis and stage. Our data indicate that NEDD4‐1 overexpression is a feature of both colorectal and gastric carcinomas. The increased expression of NEDD4‐1 in malignant gastric and colorectal cells compared to their normal epithelial cells suggests that NEDD4‐1 expression may play a role in colorectal and gastric cancer development.

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.

Increased expression of Gab2, a scaffolding adaptor of the tyrosine kinase signalling, in gastric carcinomas.

Aims: Mounting evidence indicates that alterations of protein kinase signalling pathways play crucial roles in the pathogenesis of cancers. Gab2 (Grb2‐associated binding protein 2), a member of the family of Gab scaffolding adaptors, transmits and amplifies the signals from receptor tyrosine kinases. A recent study demonstrated that Gab protein was over‐expressed in breast cancers, and the over‐expressed Gab2 increased proliferation and invasion of the cells, indicating that Gab2 is an oncogenic protein. However, the roles of Gab in other cancers are largely unknown. Methods: In this study, to see whether Gab2 expression could be a characteristic of gastric cancers, we analysed the expression of Gab2 in 60 gastric adenocarcinomas by immunohistochemistry using a tissue microarray. Results: In the normal gastric mucosal epithelial cells, Gab2 protein was expressed in parietal and zymogen cells, but not in other mucosal epithelial cells. In the cancer cells, Gab2 expression was detected in 40 (67%) of the 60 gastric adenocarcinomas. The Gab2 expression was observed in 12 (60%) of the 20 early gastric carcinomas and 28 (70%) of the 40 advanced gastric carcinomas. There was no significant association of Gab2 expression with clinocopathological characteristics, including invasion, metastasis and stage. Conclusion: Our data indicate that Gab2 over‐expression is a feature not only of breast cancers, but also of gastric cancers. Increased expression of Gab2 in malignant gastric cells compared with normal epithelial cells suggests that Gab2 expression may play a role in gastric cancer development.

Decreased expression of Bax-interacting factor-1 (Bif-1) in invasive urinary bladder and gallbladder cancers

Aims: Mounting evidence indicates that deregulation of apoptosis is involved in the mechanisms of cancer development. Bax‐interacting factor‐1 (Bif‐1) interacts with both Bax and Bak that are crucial for the intrinsic apoptosis signalling. Functionally, loss of Bif‐1 expression has been proven to enhance tumorigenesis. The aim of this study was to explore whether loss of Bif‐1 expression occurs in urinary bladder (UB) and gallbladder (GB) cancer tissues. Methods: We analysed Bif‐1 protein expression in 41 transitional cell carcinomas of UB and 26 GB adenocarcinomas by immunohistochemistry. Results: In both UB and GB, normal mucosal epithelial cells strongly expressed Bif‐1 protein. In the UB cancers, Bif‐1 expression was strongly positive in 25 cases (61.0%), but the remaining 16 cases showed no (14.6%) or markedly decreased (24.4%) Bif‐1 immunostaining compared with the normal mucosal epithelial cells. Similarly, in the GB cancers, Bif‐1 immunostaining was strong in 17 cases (65.4%), while the remaining nine cases showed no (15.4%) or markedly decreased (19.2%) Bif‐1 immunostaining compared with the normal mucosal epithelial cells. Conclusion: The decreased expression of Bif‐1 in large fractions of both UB and GB cancers (39.0% and 34.6%, respectively) compared with their normal mucosal cells suggested that loss of Bif‐1 expression might play a role in tumorigenesis in both UB and GB cancers, possibly by inhibiting apoptosis mediated by Bif‐1.