Zhang Cao

Down-regulation of ATBF1 is a major inactivating mechanism in hepatocellular carcinoma

Aims:  α‐Fetoprotein (AFP) is frequently detected in hepatocellular carcinomas (HCCs) and AT motif binding factor 1 (ATBF1) down‐regulates AFP gene expression in hepatic cells. The ATBF1 gene also inhibits cell growth and differentiation, and altered gene expression is associated with malignant transformation. The aim was to investigate the potential role of the ATBF1 gene in HCCs.

Activation-Induced Cytidine Deaminase Expression in Gastric Cancer

Helicobacter pylori increases the risk of gastric cancer development and triggers aberrant expression of activation-induced cytidine deaminase (AID). The goal of the present study was to investigate whether AID expression is involved in the development or progression of gastric cancer and the nuclear expression of p53 protein in cancer cells. We examined the expression pattern of the AID and p53 proteins in 186 gastric adenocarcinomas by immunohistochemistry. In order to investigate the molecular mechanism of AID expression, we also searched for mutations in the AID gene by single-strand conformational polymorphism and sequencing methods. In 186 sporadic gastric cancers, AID expression was detected in the 73 corresponding normal gastric mucosa and in 50 gastric cancers. Statistically, the expression of AID protein was not associated with clinicopathological parameters, including tumor size, location, differentiation and lymph node metastasis (p > 0.05). Interestingly, a significant association was observed between AID and the nuclear expression of p53 (p = 0.0094). Mutational analysis revealed no mutation in the AID gene in the gastric cancers. These results suggest that aberrant expression of the AID protein may contribute to the development of gastric cancers and induce p53 nuclear expression.

Genetic alterations and expression pattern of CEACAM1 in colorectal adenomas and cancers.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed on epithelial cells throughout the intestinal tract and is a negative regulator of tumor cell growth, suggesting that it may function as a tumor suppressor. In this study, to determine whether the CEACAM1 is involved in colorectal tumorigenesis, we have investigated the genetic alterations, including mutations and allelic loss, of the CEACAM1 gene in 17 colonic adenomas and 123 sporadic colorectal cancers. In addition, the expression pattern of the CEACAM1 protein was examined in 60 colonic adenomas and 123 sporadic colorectal adenocarcinomas. No mutation was found in colonic adenomas, but four somatic missense mutations, L36F, T312I, V398I and A445V, were detected in colorectal cancers. Interestingly, all of the mutations were found in left-side colon cancers of the patients with clinical stage III. In LOH analysis, nine adenomas were informative for at least one of the markers and five (55.6%) showed allelic loss. Thirty-eight cancers were informative at D19S211 and D19S872 markers and 21 (56.3%) showed LOH at these markers. Statistically, the frequency of allelic loss at the CEACAM1 locus was not associated with clinicopathologic parameters (P > 0.05). In immunohistochemical analysis, loss of expression of CEACAM1 protein was detected in nine (15.0%) and 30 (24.4%) of 60 colorectal adenomas and 123 colorectal cancers. Statistically, there was no significant relationship between loss of CEACAM1 expression and clinicopathologic parameters, including clinical stage, tumor location, tumor size, lymph node metastasis and 5-year survival (P > 0.05). These data suggest that genetic alteration and loss of expression of the CEACAM1 may contribute to the development of colorectal cancers, as an early event.

Genetic alterations of the Cdx2 gene in gastric cancer.

Gastric atrophy and intestinal metaplasia are generally considered precancerous lesions of the stomach; Cdx2 plays an important role in intestinal metaplasia and gastric carcinogenesis. To elucidate the potential etiological role of the Cdx2 gene in gastric carcinogenesis, we analyzed genetic mutations and allelic loss in the Cdx2 gene of 95 sporadic gastric cancers. We found two somatic missense mutations in the Cdx2 gene, P63L in exon 1 and E204K in exon 2, encoding the caudal‐like protein activation region (codon 13–180) and the homeobox domain (codon 188–243) of the gene, in the gastric cancers. In addition, 9 (25.0%) of 36 informative cases showed allelic loss at D13S220 and/or D13S260. In 11 cases with a genetic alteration, Cdx2 nuclear staining was observed only in 8 cases of gastric mucosa with intestinal metaplasia. Loss or reduced expression of the Cdx2 gene in cancer cells was found in two cases with a somatic mutation and in three cases with LOH. Interestingly, all of the cases were intestinal‐type gastric cancers. Thus, these results suggest that genetic alterations of the Cdx2 gene may contribute to the loss of Cdx2 expression and to the development of gastric cancer, especially in the intestinal‐type.

Genetic and epigenetic analysis of the VHL gene in gastric cancers

The von Hippel-Lindau tumor suppressor gene (VHL), which is located on chromosome 3p25, plays an important role in tumorigenesis, particularly in tumor growth and vascularization. Mutations of the VHL gene have been observed in the hereditary VHL syndrome and a variety of other sporadic cancers. In this study, in order to investigate whether the VHL gene is involved in gastric carcinogenesis, we have examined the genetic alterations, including somatic mutations and allelic loss, with the two microsatellite markers, D3S1038 and D3S1110, as well as promoter hypermethylation of the VHL gene in 88 sporadic gastric adenocarcinomas. No mutation was detected in the coding region of the VHL gene. Allelic loss was found in 20 (33.9%) of 59 informative cancer cases at one or both markers. In addition, promoter hypermethylation was not detected in the gastric cancer samples. This is the first investigation of the genetic and epigenetic alterations of the VHL gene in gastric cancers. Our results suggest that genetic and epigenetic alterations of the VHL gene may be not involved in the development or progression of gastric cancers. The findings also provide evidence for the presence of another gastric cancer specific tumor suppressor gene at the 3p25 region.

Analysis of succinate dehydrogenase subunit B gene alterations in gastric cancers.

Recently, the succinate dehydrogenase subunit B gene, SDHB, has emerged as a novel tumor suppressor. In this study, we have examined the genetic and epigenetic alterations of the SDHB gene in sporadic gastric adenocarcinomas in order to investigate if the SDHB gene is involved in gastric carcinogenesis. The expression of SDHB proteins was also examined with immunohistochemistry and Western blot in 184 and eight gastric cancers, respectively. There was loss or reduced expression of SDHB in 45 (24.5%) of the 184 gastric cancers. Statistically, altered expression of SDHB was not associated with clinicopathological parameters, including tumor differentiation, location, depth of invasion, and lymph node metastasis (P > 0.05). Western blot analysis showed a reduced expression of SDHB in four (50.0%) of the eight paired gastric cancer tissues. Genetic analysis showed one missense mutation, GCC → ACC (Ala → Thr) at codon 29. In addition, promoter hypermethylation was not detected in the gastric cancer samples. This is the first investigation of the genetic and protein expression analysis of the SDHB gene in gastric cancers. Our results suggest that genetic, epigenetic, and protein expression pattern alterations of the SDHB gene might play a minor role in the development or progression of gastric cancers.

Absence of E17K mutation in the pleckstrin homology domain of AKT1 in gastrointestinal and liver cancers in the Korean population

AKT/PKB (protein kinase B) kinases regulate diverse cellular processes, including cell proliferation and survival, tumor cell invasion and angiogenesis (1). Growth-factor-induced AKT/ PKB activation is mediated by phosphatidylinositol-3 kinase (PI3K), which in turn activates phosphoinositide-dependent kinase (PDK)1 and PDK2 by the production of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3, 4,5)P3) or phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) lipids (2, 3). The lipids also recruit AKT to the plasma membrane by means of its pleckstrin homology (PH) domain, and AKT is subsequently phosphorylated, resulting in full activation (3, 4). Several lines of evidence suggest that components of the AKT pathway are involved in the oncogenic transformation of normal cells. First, amplification and frequent somatic mutations of the PI3K catalytic subunit have been identified in ovarian, breast and colon cancers (5–7). In addition, PI3K is a potent transforming gene in cultured chicken embryo fibroblasts and induces activation of AKT activity (8). Second, AKT fused to the membrane-targeting gag sequence is able to transform fibroblasts (9) and activated AKT is a well-established survival factor, exerting anti-apoptotic activity by preventing release of cytochrome c from mitochondria (10). AKT1 kinase activity is often increased in prostate and breast cancers, and is associated with a poor prognosis (11). Furthermore, an activating somatic mutation (E17K) in the PH domain of the PKB/AKT gene, which results in an increased level of AKT phosphorylation, has also been identified in breast, colorectal and ovarian cancers (12). Third, a negative regu-

Mutational analysis of the HIPK2 gene in keratoacanthoma and squamous cell carcinoma of the skin

Homeodomain interacting protein kinase (HIPK) contains three distinct members that regulate apoptosis, cell growth, and proliferation. Of these, HIPK2 is a nuclear protein localized in the nuclear bodies and is prominently involved in two basic cellular functions: regulation of cell cycle ⁄apoptosis and steering of transcription (1). HIPK2 binds tumour suppressor p53 and regulates its localization, phosphorylation, acetylation, and transcriptional activity (2–4). In addition, DNA damage-inducing agents activate HIPK2 to induce p53-mediated apoptosis (5). A recent research showed HIPK2 knock-out mice rapidly develop carcinomas in situ and invasive squamous cell carcinomas (SCCs) of the skin (6). Taken together, these findings suggest that HIPK2 function as a tumour suppressor in the mouse skin. Keratoacanthomas (KAs) are common skin tumours, which are considered to be a benign variant of SCCs with the propensity to grow rapidly and regress spontaneously (7). They have been associated with exposure to ultraviolet radiation and thermal burn injuries. To the best our knowledge, mutations of the HIPK2 gene have not been described in KAs and SCCs of the skin. To analyze whether the genetic alterations of the HIPK2 gene contribute to the development of KAs and SCCs, we searched for somatic mutations of the HIPK2 gene in KAs and SCCs of the skin. Forty-three KAs and 90 SCCs between 2003 and 2004 were evaluated. Approval was obtained from the institutional review board of the Catholic University of Korea, College of Medicine. The tumour lesions and surrounding normal squamous cells were selectively obtained from haematoxylin and eosin-stained slides using a laser microdissection device (ION LMD; JungWoo International Co., Seoul, Korea). DNA extraction was performed by a modified single-step DNA extraction method (8). Genomic DNAs were amplified with primers covering the coding region of the HIPK2 gene (Table 1). The numbering of HIPK2 DNA was carried out according to the genomic sequence of the GenBank accession no. NM_022740. All PCR products in exons 3–14 of the HIPK2 gene were screened by singlestrand conformation polymorphism (SSCP) analysis (Mutation Detection Enhancement; FMC BioProducts, Rockland, ME, USA) with 10% glycerol and sequenced on the ABI prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The experiments were repeated three times to ensure specificity of the results and the consistency of data. Finally, we found 2 (4.6%) and 2 (2.2%) mutations in 43 KA and 90 SCC cases, respectively. As shown in Fig. 1, two missense mutations in KAs, P420L and A642V, were detected in exon 6 located within the kinase domain and in exon 8 located within homeoprotein interaction domain, respectively. These domains are associated with HIPK2-regulated apoptosis by mediating down-regulation of anti-apoptotic genes (4). Thus, it is likely that the HIPK2 mutations detected in this study may contribute to the development of KA via inactivation of apoptotic function. And 2 HIPK2 mutations detected in SCCs were silent mutations. All cases showed aberrant bands of the mutant allele with those of the wild type allele in SSCP analysis, indicating a hemizygous mutation in one allele and the presence of the remaining allele. The corresponding normal samples showed no evidence of mutation by repeated SSCP, suggesting that the mutations occurred somatically. Recently, loss-of-function mutations of HIPK2 gene were found in a very small subset (1.5%) of acute myeloid leukaemia and myelodyplastic syndrome (9). We also found missense mutations in only 2 (4.6%) of 43 KAs, but not in SCCs. Thus, these results suggest that the mutations of the HIPK2 gene maybe a rare event in the development of KA and SCC of the skin in Korean population. Interestingly, KA is a controversial lesion; some regard KA as a variant of SCC or within the same spectrum (7), while others consider it to be a unique entity (10). As HIPK2 mutations were found in KA, our results further support that KA may be a variant of well-differentiated