Mutational analysis of the beta-catenin gene in gastric carcinomas.

Previous studies reported that mutation of the adenomatous polyposis coli (APC) gene was not observed in the majority of gastric cancers. To evaluate the role of the APC/beta-catenin/Tcf pathway, we analyzed mutations in the beta-catenin gene and the accumulation of beta-catenin protein in gastric carcinomas. An interstitial deletion spanning exon 3 of the beta-catenin gene was observed in 1 of 13 gastric cancer cell lines. No missense mutation was found in these 13 cell lines. Nuclear and/or cytoplasmic localization of beta-catenin was observed in 16 of 70 primary gastric carcinomas by immunohistochemistry, while we found no mutations in exon 3 in 35 carcinoma tissues available for PCR amplification. Our findings suggest that somatic mutations of the beta-catenin gene are rare in human gastric carcinomas and that accumulation of normal beta-catenin protein in a subset of gastric cancers may be due to other mechanisms of its activation.     

Mutational analysis of STK11 gene in ovarian carcinomas.

Recently STK11, the causative gene of Peutz-Jeghers syndrome (PJS) was identified on chromosome 19p13.3. PJS is often accompanied by several malignancies, including breast tumor, adenoma malignum of the uterine cervix, and ovarian tumor. To investigate the involvement of STK11 gene in the development of ovarian carcinomas, we analyzed 30 ovarian carcinomas for loss of heterozygosity (LOH) and STK11 gene mutations. We found one missense mutation (codon 281, Pro to Leu) with heterozygous and somatic status. This mutation occurred at codon 281, which lies within the mutational hot spot (codon 279-281) of STK11 gene previously reported in PJS. We also detected LOH in 2 (11%) of 19 informative ovarian carcinomas. Our results suggest that mutations of the STK11 gene may play a limited role in the development of ovarian carcinomas.     

Mutational analysis of the CTNNB1 (beta-catenin) gene in human endometrial cancer: frequent mutations at codon 34 that cause nuclear accumulation

Recently, CTNNB1 (beta-catenin) has been found to function as an oncoprotein that works in the Wnt signaling pathway, and mutation of this gene has been reported in various human cancers. In this study, we analyzed 44 endometrial cancers and found somatic missense mutations in five (11%) tumors. Interestingly, four (80%) of the five tumors with mutations would cause amino acid alterations at residues next to Ser 33, one of the targets for phosphorylation of glycogen synthase kinase (GSK)-3beta. The tumors with mutations showed accumulation of the CTNNB1 protein in cytoplasm and nucleus. This is the first report of frequent somatic mutation of the CTNNB1 gene at codons adjacent to those encoding to Ser/Thr residues in endometrial cancer.     

Mutational analysis of the hCDC4 gene in gastric carcinomas

hCDC4, a ubiquitin ligase, plays a role in the control of cell cycle and chromosome stability. The hCDC4 gene is considered a tumour suppressor gene and is mutated in several human neoplasias, including colorectal and endometrial tumours. Data on the hCDC4 mutation in gastric cancer is, however, lacking. This study explored the possibility that hCDC4 mutation is involved in the development of gastric cancer. The hCDC4 gene in 162 gastric adenocarcinoma tissues was analysed for somatic mutations using a polymerase chain reaction-single strand conformation polymorphism assay. Overall, six hCDC4 mutations were found (3.7%), comprising four missense, one frameshift deletion and one nonsense mutation(s). It is notable that the hCDC4 mutations were found in early as well as in advanced gastric carcinomas. These data indicate that hCDC4 mutation occasionally occurs in gastric carcinomas and suggest that it might play a role in the development of some gastric carcinomas.     

Mutations of the beta-catenin gene in endometrial carcinomas.

To investigate the contribution of beta-catenin to the development of endometrial carcinoma, we searched for genetic alterations of the beta-catenin gene in primary endometrial carcinomas. Mutational analysis of exon 3 of the beta-catenin gene, encoding the serine/threonine residues for GSK-3 beta phosphorylation, was performed for 35 tumors. Nucleotide sequencing analysis revealed that 5 tumors (5/35, 14%) contained mutations (S33C, S37C, S37F, T41A) that altered potential GSK-3 beta phosphorylation sites. Each of the mutations resulted in the substitution of serine/threonine residues that have been implicated in the down-regulation of beta-catenin through phosphorylation by GSK-3 beta kinase. Furthermore, the incidence of beta-catenin mutations was significantly higher in early-onset (3 of 5) than that in late-onset tumors (2 of 30) (P = 0.014, Fisher's exact test). Replication error (RER)-positive phenotype was not detected in tumors with the beta-catenin gene mutation, although 10 of 35 tumors revealed RER. We performed immunohistochemistry of beta-catenin in 17 cases for which tissue samples were available. We confirmed accumulation of beta-catenin protein in both the nucleus and cytoplasm in 3 tumors, including two in which amino acid alterations had occurred at codon 33 and 37. The other case had no mutation in exon 3. Our results suggested that mutations at serine/threonine residues involved in phosphorylation by GSK-3 beta affected the stability of beta-catenin. Accumulation of mutant beta-catenin could contribute to the development of a subset of endometrial carcinomas, particularly those of the early-onset type.     

Mutational analysis of BRCA1 and BRCA2 and clinicopathologic analysis of ovarian cancer in 82 ovarian cancer families: two common founder mutations of BRCA1 in Japanese population.

We analyzed genetic alterations in BRCA1 and BRCA2 genes among 82 ovarian cancer families in Japan. The clinical characteristics of BRCA-associated ovarian cancer patients were compared with cases carrying no mutations as well as with population controls. Using a direct sequencing method, 45 of the 82 ovarian cancer families were found to carry BRCA1 or BRCA2 germ-line mutations (40 with BRCA1 and 5 with BRCA2). In 24 independent mutations of BRCA1, 5 recurrent mutations were found and 2 of them, the L63X and Q934X mutations, were detected in seven and eight independent families, respectively. In addition, 16 mutations of BRCA1 and 3 mutations of BRCA2 have never been described previously. In consideration of clinicopathological features, there was a significantly higher proportion of tumors with serous adenocarcinoma and of cases of advanced stages in the BRCA1 or BRCA2 cases than in those of the controls. On the other hand, there were no differences of mean age at diagnosis between patients with BRCA1 or BRCA2 mutation and those of the controls. Our results indicate that the features of BRCA-associated ovarian cancer in Japan appear to be similar to those in Western countries, and the L63X and Q934X mutations of BRCA1 appear to be common founder mutations unique to the Japanese population.     

Frequent somatic mutations of the beta-catenin gene in intestinal-type gastric cancer.

The increased level of cytoplasmic beta-catenin through the mutations to either beta-catenin or adenomatous polyposis coli (APC) has been proposed as an important oncogenic step in various tumors. Gastric cancer showed frequent genetic alterations of the APC gene, and the risk for gastric cancer in familial adenomatosus polyposis patients is 10 times higher than that in the general population. These findings raise the possibility that mutations of beta-catenin may also be associated with the development of gastric cancer. We detected seven somatic mutations in a portion of exon 3 encoding for the glycogen synthase kinase 3beta phosphorylation consensus region of the beta-catenin gene in 43 gastric cancers. All of these mutations were missense mutations, of which five are in the highly conserved aspartic acid 32 and two are in serine 29; all of these seven mutations were detected exclusively in intestinal-type gastric cancers (7 of 26; 26.9%), but not in the diffuse-type (0 of 17). We concluded that disruption of the APC/beta-catenin/T cell factor-lymphoid enhancer binding factor pathway might play an important role especially in the development of intestinal-type gastric cancer.     

Coordinate expression of Cdc25B and ER-alpha is frequent in low-grade endometrioid endometrial carcinoma but uncommon in high-grade endometrioid and nonendometrioid carcinomas

Overexpression of cdc25B, an important cell cycle regulator, has been shown to result in mammary gland hyperplasia in transgenic mice and to increase steroid hormone responsiveness as a direct coactivator of the estrogen receptor (ER). We investigated the potential role of cdc25B in the pathogenesis of endometrial carcinomas in conjunction with ER-alpha. We examined the expression of cdc25B and phosphorylated ER-alpha in 4 archived human specimens of normal endometrium; 7 endometrial hyperplasia with or without atypia; 32 endometrioid endometrial carcinoma (EEC), including 20 low-grade (grade 1) and 12 high-grade (grade 2 or 3) tumors; and 18 endometrial cancers with aggressive histological subtypes (uterine papillary serous carcinoma and clear cell carcinoma, UPSC/CCC) by immunohistochemistry with monoclonal antibodies. Expression of cdc25B and phosphorylated ER-alpha was increased in endometrial hyperplasia and atypical hyperplasia compared with normal secretory endometrium. Ninety percent (18 of 20) of the low-grade EEC expressed cdc25B at a high level, whereas only 42% (5 of 12) of the high-grade EEC did so (chi(2) = 8.7; P < 0.01). Sixty-five percent (13 of 20) of the low-grade EEC expressed phosphorylated ER-alpha at high levels, but only 17% (2 of 12) of high-grade EEC did so (chi(2) = 7.0; P < 0.01). Coordinate high-level expression of phosphorylated ER-alpha and cdc25B occurred in 65% (13 of 20) of low-grade EEC but in only 17% (2 of 12) of the high-grade EEC (chi(2) = 7.0; P < 0.01). In the UPSC/CCC tumors, only 22% (4 of 18) of the tumors expressed phosphorylated ER-alpha at high-levels. However, 83% (15 of 18) of these carcinomas showed high expression of cdc25B (chi(2) = 13.5; P < 0.01). The majority of the UPSC/CCC (15 of 18) did not show coordinate high expression of phosphorylated ER-alpha and cdc25B. Our findings show that in endometrial hyperplasia and low-grade EEC, coordinate increase in cdc25B and phosphorylated ER-alpha occurs. However, in UPSC/CCC, cdc25B is highly expressed without coordinate increase in phosphorylated ER-alpha. Cdc25B may play important roles in the development and progression of EEC and UPSC/CCC by different mechanisms.     

Evidence for divergence of DNA copy number changes in serous, mucinous and endometrioid ovarian carcinomas.

Comparative genomic hybridization was applied to detect and map changes in DNA copy number in 24 well or moderately differentiated epithelial ovarian carcinomas (eight serous, eight mucinous and eight endometrioid carcinomas). Twenty-three of the 24 tumours showed changes in DNA copy number in one or several regions (median 4, range 1-17). Gains were more frequent than losses (ratio 1.6:1.0). The most frequent gains occurred in chromosomes 1q (38%), 2p (29%), 7q (25%), 8q(38%) and 17q (38%), and the most common losses were located in chromosomes 8p (21%), 9p (25%) and 13q (21%). High-level amplifications were detected in seven tumours at 1q22-32, 2p15-22, 3qcen-23, 6p21-22 and 8q. In the three histological subtypes the copy number karyotypes showed substantial differences. Gains at 1q were observed in endometrioid (five cases) and serous tumours (four cases). Increased copy number at 10q was seen in endometrioid tumours only (four cases), whereas gains at 11q occurred mostly in serous tumours (four cases). In mucinous tumours, the most common copy number change was a gain at 17q (six cases). The results show that, in epithelial ovarian carcinoma, changes in DNA copy number are a rule rather than an exception, chromosomes 1, 2, 7, 8, 9, 13 and 17 being the most frequently affected. The diverging pattern of genetic changes seen in epithelial ovarian carcinomas with different histological phenotypes suggests that various pathways may lead to tumorigenesis and/or progression in these subgroups.     

Mutational spectrum of beta-catenin,AXIN1, and AXIN2 in hepatocellular carcinomas and hepatoblastomas

Activation of Wnt signaling through beta-catenin mutations contributes to the development of hepatocellular carcinoma (HCC) and hepatoblastoma (HB). To explore the contribution of additional Wnt pathway molecules to hepatocarcinogenesis, we examined beta-catenin, AXIN1 and AXIN2 mutations in 73 HCCs and 27 HBs. beta-catenin mutations were detected in 19.2% (14 out of 73) HCCs and 70.4% (19 out of 27) HBs. beta-catenin mutations in HCCs were primarily point mutations, whereas more than half of the HBs had deletions. AXIN1 mutations occurred in seven (9.6%) HCCs and two (7.4%) HBs. The AXIN1 mutations included seven missense mutations, a 1 bp deletion, and a 12 bp insertion. The predominance of missense mutations found in the AXIN1 gene is different from the small deletions or nonsense mutations described previously. Loss of heterozygosity at the AXIN1 locus was present in four of five informative HCCs with AXIN1 mutations, suggesting a tumor suppressor function of this gene. AXIN2 mutations were found in two (2.7%) HCCs but not in HBs. Two HCCs had both AXIN1 and beta-catenin mutations, and one HCC had both AXIN2 and beta-catenin mutations. About half the HCCs with AXIN1 or AXIN2 mutations showed beta-catenin accumulation in the nucleus, cytoplasm or membrane. Overall, these data indicate that besides the approximately 20% of HCCs and 80% of HBs with beta-catenin mutations contributing to hepatocarcinogenesis, AXIN1 and AXIN2 mutations appear to be important in an additional 10% of HCCs and HBs.     

beta-catenin mutations are absent in hepatocellular carcinomas of SV40 T-antigen transgenic mice

beta-catenin mutations have been found not only in melanoma and prostatic carcinoma but also in hepatocellular carcinomas in human, c-myc, H-ras genes transgenic mice and chemically-induced models. We investigated beta-catenin mutations in human hepatocellular carcinomas (HCCs), Hep G2 cell line and HCCs in SV40 T-antigen transgenic mice, in order to examine whether beta-catenin mutations are frequently observed in HCC in general. We found a point mutation of beta-catenin in one of nine HCCs in human and a deletion of it in Hep G2 cell line. However, we found no mutation in HCC in SV40 TG mice liver.     

Hepatocarcinogenesis in mice with beta-catenin and Ha-ras gene mutations

We have established previously a mouse strain containing a mutant beta-catenin allele of which exon 3 was sandwiched by loxP sequences [Catnb(lox(ex3))]. In this mouse strain, a Wnt-activating beta-catenin mutation alone is insufficient for hepatocarcinogenesis, but additional mutations or epigenetic changes may be required. Here we report that hepatocellular carcinoma develops at the 100% incidence in mice with simultaneous mutations in the beta-catenin and H-ras genes that are introduced by adenovirus-mediated Cre expression. Although H-ras mutation alone rapidly causes large cell dysplasia in the hepatocytes, these cells show no autonomous growth within 1 week after infection of the Cre-adenovirus. However, simultaneous induction of an additional mutation in the beta-catenin gene causes a clonal expansion of such dysplastic cells, followed by nodular formation and development of hepatocellular carcinoma. These results indicate that beta-catenin mutations play a critical role in hepatocarcinogenesis in cooperation with another oncogene and that these mice provide a convenient model to investigate early steps of hepatocarcinogenesis.     

Accumulation of beta-catenin protein and mutations in exon 3 of beta-catenin gene in gastrointestinal carcinoid tumor

The molecular basis of carcinogenesis in gastrointestinal carcinoid tumors is not well understood. To clarify the contribution of the Wnt/beta-catenin signaling to this type of carcinogenesis, we investigated 72 cases of gastrointestinal carcinoid tumor both immunohistochemically and by direct sequencing of beta-catenin. Accumulation of beta-catenin in the cytoplasm and/or nucleus was observed in 57 cases (79.2%). We also detected mutations in exon 3 of beta-catenin in 27 cases (37.5%) and one mutation in APC (1.4%). Our results suggest that alterations in the Wnt/beta-catenin signaling pathway may be involved in the development of gastrointestinal carcinoid tumors.     

Absence of p53 gene mutations in primary nasopharyngeal carcinomas.

Alterations in the p53 tumor suppressor gene and Epstein-Barr virus status were investigated in 15 nasopharyngeal carcinoma (NPC) biopsies, 4 xenografts, and 2 cell lines from the Cantonese region of southern China. One other established NPC cell line obtained from a northern Chinese patient was also studied. Restriction fragment length polymorphism analysis revealed a loss of heterozygosity for chromosome 17p, where the p53 gene resides, in only one of 15 NPC biopsies. Polymerase chain reaction-single-stranded conformational polymorphism analysis and direct sequencing failed to detect sequence alterations in exons 5 through 8 of the p53 gene in the 15 tumors and in the 4 NPC xenografts, all of which tested positive for Epstein-Barr virus. In contrast, the 3 NPC cell lines were all negative for Epstein-Barr virus and contained G----C transversions in the p53 gene, with cell lines CNE-1 and CNE-2 harboring identical AGA (arginine) to ACA (threonine) changes at codon 280. These results suggest that p53 inactivation is not a necessary component of nasopharyngeal carcinogenesis in Cantonese but may be important in the establishment of cell lines derived from these tumors.