Sang Ho Kim

Somatic Mutations of ERBB2 Kinase Domain in Gastric, Colorectal, and Breast Carcinomas

Purpose: Recent reports revealed that the kinase domain of the ERBB2 gene is somatically mutated in lung adenocarcinoma, suggesting the mutated ERBB2 gene as an oncogene in human cancers. However, because previous reports focused the mutational search of ERBB2 primarily on lung cancers, the data on ERBB2 mutations in other types of human cancers have been largely unknown. Experimental Design: Here, we did a mutational analysis of the ERBB2 kinase domain by PCR single-strand conformational polymorphism assay in gastric, colorectal, and breast carcinoma tissues. Results: We detected the ERBB2 kinase domain mutations in 9 of 180 gastric carcinomas (5.0%), in 3 of 104 colorectal carcinomas (2.9%), and in 4 of 94 breast carcinomas (4.3%). All of the detected ERBB2 mutations except for one in-frame deletion mutation were missense mutations. Of the 16 ERBB2 mutations detected, 4 affected Val777 in the exon 20 site, and 3 affected Leu755 in the exon 19 site. We simultaneously analyzed the somatic mutations of EGFR, K-RAS, PIK3CA, and BRAF genes in the 16 samples with ERBB2 mutations, and found that all of the 3 colorectal carcinoma samples with ERBB2 mutations harbored K-RAS mutations. Conclusion: This study showed that in addition to lung adenocarcinomas, ERBB2 kinase domain mutation occurs in other common human cancers such as gastric, breast, and colorectal cancers, and suggested that alterations of ERBB2-mediated signaling pathway by ERBB2 mutations alone or together with K-RAS mutations may contribute to the development of human cancers.

Caspase-8 gene is frequently inactivated by the frameshift somatic mutation 1225_1226delTG in hepatocellular carcinomas.

Evidence exists that alterations of the genes encoding apoptosis-related proteins contribute to either development or progression of human cancers. Caspase-8 plays a crucial role in the initiation phase of apoptosis. To explore the possibility that the genetic alteration of caspase-8 gene is involved in the development of hepatocellular carcinomas (HCCs), we have analysed the entire coding region of human caspase-8 gene for the detection of somatic mutations by polymerase chain reaction-single-strand conformation polymorphism in 69 HCCs with low-grade dysplastic nodule (LGDN, n=2) or high-grade dysplastic nodule (HGDN, n=2) or without any dysplastic nodules (n=65). Overall, we detected a total of nine somatic mutations in 69 HCCs (13.0%). Interestingly, all of the nine mutations were an identical frameshift mutation with two base-pair deletion (1225_1226delTG), which would result in a premature termination of amino-acid synthesis in the p10 protease subunit. In a patient sample, we detected the 1225_1226delTG mutation both in HCC and LDGN lesions, suggesting that caspase-8 mutation could be involved in the early stage of HCC carcinogenesis. We expressed the tumor-derived caspase-8 mutant in the cells and found that the mutant abolished cell death activity of caspase-8. Our data indicate that caspase-8 gene is frequently mutated in HCC and the majority of the mutations may be the frameshift mutation 1225_1226delTG. Also, the data suggest that caspase-8 gene mutation might lead to the loss of its cell death function and contribute to the pathogenesis of HCC.

Inactivating mutations of CASPASE-7 gene in human cancers.

Caspase-7 is a caspase involved in the execution phase of apoptosis. To explore the possibility that the genetic alterations of CASPASE-7 might be involved in the development of human cancers, we analysed the entire coding region and all splice sites of human CASPASE-7 gene for the detection of somatic mutations in a series of human solid cancers, including carcinomas from stomach, colon, head/neck, esophagus, urinary bladder and lung. Overall, we detected CASPASE-7 mutations in two of 98 colon carcinomas (2.0%), one of 50 esophageal carcinomas (2.0%) and one of 33 head/neck carcinomas (3.0%). We expressed the tumor-derived caspase-7 mutants in 293 T cells and found that the apoptosis was reduced compared to the wild-type caspase-7. This is the first report on the CASPASE-7 gene mutations in human malignancies, and our data suggest that the inactivating mutations of the CASPASE-7 gene might lead to the loss of its apoptotic function and contribute to the pathogenesis of some human solid cancers.

Mutational analysis of the ARAF gene in human cancers

Deregulation of RAS signal transduction has been implicated in the malignant growth of human cancer cells. The BRAF gene, encoding a RAF family member in the downstream pathway of RAS, is somatically mutated in a number of human cancers, raising the possibility that other RAF family members might be mutated in human cancers. In this study we analyzed the genomic DNAs for the detection of somatic mutations of the ARAF gene in 60 human cancer cell lines and 323 primary human cancer tissues, including colorectal carcinomas, gastric carcinomas, ovarian tumors and acute leukemias. The MOLT‐4 leukemia cell line was found to harbor an ARAF gene mutation resulting in an amino acid substitution (A451T) at the activation segment in the kinase domain of ARAF. In the cancer tissues we could not detect any ARAF gene mutation. Our data indicate that, in contrast to the BRAF gene, the ARAF gene is rarely mutated in human cancers, and suggest that alterations of the RAS pathway by ARAF gene mutation may not play an important role in the pathogenesis of human cancers.

Mutational analysis of the CASP6 gene in colorectal and gastric carcinomas

Failure of apoptosis is one of the hallmarks of cancer. As an execution‐phase caspase, caspase‐6 plays a crucial role during apoptosis. To explore the possibility that the genetic alterations of CASP, which encodes caspase‐6, might be involved in the development of human cancers, we analyzed the entire coding region and all splice sites of the human CASP6 gene for the detection of somatic mutations in 100 colorectal carcinomas and 50 gastric carcinomas. Overall, we detected three somatic mutations of the CASP6 gene, including two missense mutations and one splice‐site mutation. The mutations were observed in two of the 100 colorectal carcinomas (2.0%) and one of the 50 gastric carcinomas (2.0%). Of note, one colorectal carcinoma with the CASP6 mutation harbored CASP3 and CASP8 gene mutations as well. We also analyzed caspase‐6 expression by immunohistochemistry, and found that caspase‐6 was expressed in 60% of the gastric cancers and 90% of the colorectal cancers. This is the first report on CASP6 gene mutations in human cancers, and these data indicate that the CASP6 gene is occasionally mutated in gastric and colorectal carcinomas. Also, the data suggest the possibility that deficiency of caspase‐6 expression might contribute to the pathogenesis of gastric cancers.