Jong Heun Lee

Increased expression of histone deacetylase 2 is found in human gastric cancer.

Accumulated evidence has established that aberrant regulation of histone deacetylases (HDACs) is one of the major causes of the development of human malignancies. Among different iso‐enzymes of HDAC and sirtuins grouped as the HDAC super family, little is known as to how histone deacetylase 2 (HDAC2) causes carcinogenesis in solid tumors. Here, in order to investigate the possible role of HDAC2 in gastric carcinogenesis, we analyzed the expression of HDAC2 in 71 gastric adenocarcinomas by immunohistochemistry. Moderate to strong expression of HDAC2 was found in 44 (62%) out of a total of 71 tumors. The majority of positive tumors, which were detected in the nucleus but not in normal gastric epithelium, did not express HDAC2 or showed only weak positive staining. Interestingly, we also noted that HDAC2 expression appeared to be associated with tumor aggressiveness as HDAC2 expression was observed to be statistically significant in advanced gastric cancer (P=0.0023, Chi‐square test) and in positive lymph node metastasis (P=0.0713, Chi‐square test). Taken together, these results suggest that HDAC2 may play an important role in the aggressiveness of gastric cancer.

Inactivating mutations of caspase-8 gene in colorectal carcinomas

BACKGROUND & AIMS There has been evidence that dysregulation of apoptosis is involved in the pathogenesis of cancer development. Caspase-8 is an initiation caspase that activates the caspase cascade during apoptosis. The aim of this study was to explore the possibility that mutation of the caspase-8 gene might be involved in the development of colorectal cancer. METHODS We analyzed the entire coding region of the caspase-8 gene for the detection of somatic mutations in 180 colorectal tumors (98 invasive carcinomas and 82 adenomas) by polymerase chain reaction, single-strand conformation polymorphism, and DNA sequencing. RESULTS Overall, we detected a total of 5 somatic mutations in 98 invasive carcinomas (5.1%), but no mutations were detected in 82 adenomas (0%). The frequency of caspase-8 mutation in the carcinomas was significantly higher than that in adenomas (P < 0.05). The 5 mutations consisted of 1 frameshift, 1 nonsense mutation, and 3 missense mutations. We expressed the 5 tumor-derived caspase-8 mutants and found that 3 of the 5 mutations markedly decreased apoptosis activity of caspase-8. Furthermore, expression of the inactivating caspase-8 mutants interfered with apoptosis by death receptor overexpression, indicating that these mutants have dominant-negative inhibition of the death receptor-induced apoptosis. CONCLUSIONS The presence of caspase-8 mutation in colon carcinomas suggests that caspase-8 gene mutation might lead to the loss of its apoptotic function and contribute to the pathogenesis of colorectal carcinomas, especially at the late stage of colorectal carcinogenesis.

Somatic mutations of TRAIL-receptor 1 and TRAIL-receptor 2 genes in non-Hodgkin's lymphoma

Tumor necrosis factor-related apoptosis-inducing ligand-receptor 1 (TRAIL-R1) and tumor necrosis factor-related apoptosis-inducing ligand-receptor 2 (TRAIL-R2) are cell-surface receptors involved in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell-death signaling. TRAIL-R1 and TRAIL-R2 genes have recently been mapped to chromosome 8p21-22, which is a frequent site of allelic deletions in many types of human tumors, including non-Hodgkins lymphoma (NHL). Because TRAIL/TRAIL receptor system plays an important role in lymphocyte homeostasis, we hypothesized that the mutations of TRAIL-R1 and TRAIL-R2 may be involved in the development of NHL and that such mutations may be responsible for the allelic losses of 8p21-22 in NHL. In this study, we analysed the entire coding region of TRAIL-R2 gene and the death domain region of TRAIL-R1 gene for the detection of the somatic mutations in a series of 117 human NHLs using polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) analysis. Overall, eight tumors (6.8%) were found to have two TRAIL-R1 gene mutations or six TRAIL-R2 gene mutations. Interestingly, of the eight mutations, six missense mutations (two TRAIL-R1 and four TRAIL-R2) were detected in the death domains and one nonsense mutation of TRAIL-R2 was detected just before the death domain. Our data suggest that somatic mutations of TRAIL-R1 and TRAIL-R2 genes may play a role in the pathogenesis of some NHLs and that TRAIL-R1 and TRAIL-R2 genes might be the relevant genes to the frequent loss of chromosome 8p21-22 in human NHL.

BRAF and KRAS mutations in stomach cancer

Ras proteins control signaling pathways that are key regulators of several aspects of normal cell growth and malignant transformation. BRAF, which encodes a RAF family member in the downstream pathway of RAS, is somatically mutated in a number of human cancers. The activating mutation of BRAF is known to play a role in tumor development. As there have been no data on the BRAF mutation in stomach cancer, we analysed the genomic DNAs from 319 stomach carcinomas for the detection of somatic mutations of BRAF. Overall, we detected BRAF mutations in seven stomach carcinomas (2.2%). Five of the seven BRAF mutations involved Val 599, the previously identified hotspot, but the substituted amino acid (V599 M) was different from the most common BRAF mutation (V599E). The remaining two mutations involved a conserved amino acid (D593G). One tumor had both BRAF and KRAS mutations. This is the first report on BRAF mutation in stomach cancer, and the data indicate that BRAF is occasionally mutated in stomach cancer, and suggest that alterations of RAS pathway both by RAS and BRAF mutations contribute to the pathogenesis of stomach cancer.

Nuclear localization of β-catenin is an important prognostic factor in hepatoblastoma

In this study, mutational and immunohistochemical analyses of β‐catenin were performed in 30 hepatoblastomas, to assess the prevalence of alterations of the Wnt pathway with respect to clinicopathological parameters and survival. Four missense mutations of β‐catenin (13.3%) were detected and there was strong immunoreactivity for β‐catenin in the cytoplasm and/or the nucleus in 97% of hepatoblastomas. Nuclear and cytoplasmic staining was demonstrated in 19 of 30 tumours (63%), while ten revealed only cytoplasmic staining. Statistically, this nuclear β‐catenin staining was significantly higher in the embryonal (Fisher exact test; p=0.00393) or undifferentiated type (p=0.00156) of hepatoblastoma than in the fetal type, but there was no difference between clinical stages I and II and clinical stages III and IV (p=0.175). Cumulative survival curves showed that nuclear β‐catenin staining (generalized Wilcoxon test; p=0.0088), undifferentiated histological type (p=0.0305), and clinical stages III and IV (p=0.0107) were significantly correlated with shorter survival time in these patients. Moreover, Cox multivariate analysis provides evidence that nuclear β‐catenin staining is the most important prognostic factor for survival (p=0.0090). It is therefore concluded that immunohistochemical analysis of β‐catenin might be a useful clinical tool for estimating the prognosis for patients with hepatoblastoma. Copyright

Inactivating mutations of the caspase-10 gene in gastric cancer.

We have analysed the genetic alteration of the entire coding region and all splice sites of caspase-8 and -10 genes in 99 gastric cancers by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) and sequencing. We found LOH of the caspase-8 and -10 in nine (28%) of 32 and in four (15%) of 26 informative cases, respectively. Overall, three of 99 gastric cancers (3%) were found to have the caspase-10 mutations, which were identified in the coding regions of the death effector domain (codon 147) and the p17 large protease domain (codons 257 and 410), whereas no mutation was detected in caspase-8. In vitro expression studies, the M147T and Q257stop mutants severely impaired caspase-10-mediated apoptosis, whereas the V410I which was the same mutation detected in ALPS patient had a significant, albeit less severe, effect on apoptosis. The data presented here suggest that somatic alterations of the caspase-10 gene might contribute to the pathogenesis in a subset of gastric cancers through the loss of their apoptotic function.

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.

Alterations of Fas-pathway genes associated with nodal metastasis in non-small cell lung cancer.

Many types of cancer cells are resistant to Fas-mediated apoptosis by several mechanisms, including the mutations of the genes involved in Fas-mediated apoptosis. In this study, to explore the possibility that the mutations of the genes involved in the proximal pathway of Fas-mediated apoptosis (Fas, FADD, caspase 8 and caspase 10) are involved in cancer metastasis, we have analysed somatic mutation and deletion of these genes in 80 non-small cell lung cancers (NSCLCs) with (n=43) and without (n=37) metastasis to the regional lymph nodes. We found 12 mutations (four Fas, four FADD, and four caspase 10 mutations) in 11 of 80 NSCLCs (13.8%). Interestingly, of these mutations, most mutations (10 out of 12) were detected in the NSCLCs with metastasis, and the frequency in the metastasis lesions (23%) was higher than that in the primary lesions of the NSCLCs without metastasis (5.4%). Furthermore, transfection study revealed that the tumor-derived mutants have decreased apoptosis inductions compared to the wild types. These data suggest that the inactivating mutations of the genes in the proximal pathway of Fas-mediated apoptosis may lead to a decreased cancer cell death and play a role in the metastasis of NSCLC.

Overexpression of S100A4 is closely related to the aggressiveness of gastric cancer

Elevated levels of the calcium‐binding protein S100A4 cause metastasis of benign rat mammary tumor cells. To investigate whether S100A4 plays an important role in the invasion and metastasis of gastric cancers, we examined the gene mutations in the coding regions and expression patterns of the S100A4 in gastric adenocarcinoma in Korea. Moderate to strong expression of S100A4 was found in 53 (68.8%) of the 77 gastric adenocarcinomas, whilst normal gastric epithelium either failed to stain or showed weak staining. Interestingly, S100A4 expression was more frequently observed in gastric cancer patients with advanced gastric cancer (p=0.039), positive lymph node metastasis (p=0.001), and peritoneal dissemination (p=0.022). No gene mutations were found in the analyzed genomic area in 77 gastric adenocarcinomas and 15 gastric cancer cell lines. We found one single nucleotide polymorphism without an amino acid change, A99G, in two cases. These data suggest that the overexpression of S100A4 may be closely related to the aggressiveness of gastric cancer in Korea.

Inactivating mutation of the pro-apoptotic gene BID in gastric cancer.

There is evidence that deregulation of apoptosis is mechanistically involved in cancer development and somatic mutations of apoptosis‐related genes have been reported in human cancers. BID, a pro‐apoptotic member of the Bcl‐2 family, interconnects the extrinsic apoptosis pathway initiated by death receptors to the intrinsic apoptosis pathway. To explore the possibility that genetic alterations of BID might be involved in the development of human cancers, this study analysed the entire coding region and all splice sites in the human BID gene in 67 advanced gastric carcinomas. Overall, four BID mutations (6.0%) were detected that consisted of one frameshift and three missense mutations. The tumour‐derived BID mutants were expressed in 293T cells and it was found that, compared with wild‐type BID, the frequency of apoptosis was significantly reduced in cells expressing the gene containing the frameshift mutation. Furthermore, expression of the inactivating frameshift mutant interfered with cell death by overexpression of death receptors, indicating that this mutant inhibits the extrinsic apoptosis pathway in a dominant‐negative fashion. Also, the frameshift mutation rendered cancer cells resistant to apoptosis induced by the anti‐cancer drug 5‐fluorouracil (5‐FU). This is the first report of BID gene mutation in human malignancy. The data suggest that such mutations occur rarely in gastric cancers and that only a small fraction of BID mutations may lead to the loss of its apoptotic function. Copyright