Frameshift mutations of OGDH, PPAT and PCCA genes in gastric and colorectal cancers.

Metabolic reprogramming is a hallmark of cancer. However, genetic alterations in metabolism-related genes are largely unknown. The aim of this study was to identify whether somatic mutations in OGDH, PPAT and PCCA genes known to be involved in amino acid or nucleotide metabolism are mutated in gastric cancer (GC) and colorectal cancer (CRC). By public database search, we identified that OGDH, PPAT and PCCA genes harbor mononucleotide repeats that may serve as mutation targets in cancers with microsatellite instability (MSI). We analyzed the repeats for the presence of the mutations in 90 GCs and 141 CRCs using single-strand conformation polymorphism (SSCP) and samples of 10 patients with shifted bands were sequenced. We found frameshift mutations of OGDH (3 cases), PCCA (5 cases) and PPAT (2 cases) in the cancers. These mutations were exclusively detected in MSI-high (MSI-H), and not in MSI-low or MSI-stable (MSI-L/MSS) cancers. We also analyzed 16 CRCs for the presence of intratumoral heterogeneity (ITH) and found that one CRC harbored regional ITH for OGDH frameshift mutation showing very rare frequency of OGDH mutation ITH in colorectal cancer tissues. Our data indicate that amino acid/nucleotide metabolism-related genes OGDH, PPAT and PCCA acquire somatic mutations in MSH-H GCs and CRCs and that mutational ITH may occur in at least some of these tumors. Collectively, our results may extend our insight into the involvement of amino acid/nucleotide metabolism in the pathogenesis of cancer for, in particular, MSI-H GCs and CRCs.

Mutational and expressional analysis of MLL genes in gastric and colorectal cancers with microsatellite instability.

MLL genes encode histone methyltransferases that are required for proper expression of a variety of genes. The pathologic implications of MLL genes have been studied not only in leukemias, but also in some solid cancers. We found in a public database that MLL, MLL2, MLL3, MLL4 and MLL5 genes had mononucleotide repeats that might be mutated in cancers with microsatellite instability (MSI). Frameshift mutations in a repeat of MLL3 have been found in colorectal cancers (CRC), but there is no frameshift mutation data of the other genes. In this study, we analyzed these repeats in 32 gastric cancers (GC) with high MSI (MSI-H), 59 GC with low MSI (MSI-L)/stable MSI (MSS), 40 CRC with MSI-H and 59 CRC with MSI-L/MSS by single-strand conformation polymorphism and DNA sequencing. We also analyzed MLL3 expression in GC and CRC tissues using immunohistochemistry. We found MLL, MLL2, MLL3 and MLL5 frameshift mutations in two (one GC and one CRC), three (one GC and two CRC), 17 (14 GC and three CRC) and six (four GC and two CRC) cancers, respectively. They were detected exclusively in MSI-H cancers, but not in MSI-L/MSS cancers. All of the cancers with MLL3 mutations showed loss of MLL3 expression, and their values were significantly lower than in those without MLL3 mutation (50.9%). Of note, the GC with MSI-H had significantly higher incidences in both MLL mutations and MLL3 expression loss than the CRC with MSI-H. Our data indicate that frameshift mutations of MLL genes and loss of expression of MLL3 protein are common in GC and CRC with MSI-H.

Mutational and expressional analyses of STAG2 gene in solid cancers.

Aneuploidy is frequently observed in cancers and is considered a crucial mechanism in cancer development. STAG2 is a gene that encodes a component of cohesion complex required for normal chromosomal segregation. Recently, somatic mutation of STAG2 gene and loss of STAG2 protein have been reported in glioblastoma, Ewing's sarcoma and melanoma. The aim of this study was to see whether such alterations of STAG2 are also common in other cancers. In this study, we analyzed STAG2 somatic mutation in 45 colorectal carcinomas (CRC), 45 gastric carcinomas (GC), 45 breast carcinomas, 45 non-small cell lung cancers and 45 prostate carcinomas (PCA) by single-strand conformation polymorphism. We analyzed also STAG2 protein expression in 100 GC, 103 CRC and 107 PCA by immunohistochemistry. STAG2 protein was well expressed in normal stomach, colon and prostate epithelial cells, while it was lost in 27% of GC, 23% of CRC and 30% of PCA. The loss of STAG2 was observed irrespective of subtypes, stages and grades of the cancers. However, we could not find any STAG2 mutations in these cancers. The loss of expression of STAG2 in GC, CRC and PCA tissues compared to their corresponding normal cells indicates that STAG2 loss is common in carcinomas as well. The data suggest also that loss of expression of STAG2, but not somatic mutation, might be responsible to STAG2 inactivation and is common in studied types of carcinomas.

Increased immunoreactivity of membrane type-1 matrix metalloproteinase (MT1-MMP) and ß-catenin in high-risk basal cell carcinoma.

BACKGROUND: Although various immunohistological markers have been investigated to assess the aggressive characteristics of basal cell carcinoma (BCC), the role of membrane type-1 matrix metalloproteinase (MT1-MMP) has not been well established. OBJECTIVES: To clarify the precise role of MT1-MMP in BCC, MT1-MMP expression was studied in various histological subtypes of BCC. MATERIALS AND METHODS: High-risk subtypes of BCC were compared by assessing the expression of ß-catenin and MT1-MMP. The tissue microarray technique was used for immunohistochemical staining. Fifty-eight samples were divided into six subtypes (10 nodular, 12 mixed, nine infiltrative, eight morphoeiform, 10 micro-nodular and nine basosquamous). Overall, the 10 nodular BCC samples were classified as low-risk BCC and the remaining 48 samples were classified as high-risk BCCs. RESULTS: ß-Catenin immunoreactivity was increased in the high-risk BCCs compared with the low-risk (nodular) BCC (P < 0·001). Nuclear ß-catenin immunoreactivity was increased at the invading front of mixed BCC tumour islands compared with the upper portion of the lesion (P < 0·01). For the mixed BCC (P < 0·01), infiltrative BCC (P < 0·001), morphoeiform BCC (P < 0·001), micronodular BCC (P < 0·001) and basosquamous (P < 0·001) carcinoma, ß-catenin immunoreactivity was increased at the invading front compared with nodular BCC. MT1-MMP immunoreactivity was increased in the high-risk BCCs compared with the low-risk (nodular) BCC (P < 0·01). The membranous MT1-MMP immunoreactivity was increased at the invading front of mixed BCC tumour islands compared with the upper portion of the lesions (P < 0·01). For the mixed (P < 0·01), infiltrative (P < 0·05), morphoeiform (P < 0·05), micronodular (P > 0·05) and basosquamous (P < 0·05) BCC, MT1-MMP immunoreactivity was also increased at the invading front compared with nodular BCC. CONCLUSIONS: The results of this study suggest that MT1-MMP might be a novel marker for high-risk BCC. In addition, expression of both ß-catenin and MT1-MMP was increased in high-risk BCC tumour cells, indicating that these two proteins may play an important role in locally invasive and highly destructive growth behaviour of high-risk BCCs.

Low frequency mutation of the Ephrin receptor A3 gene in hepatocellular carcinoma.

EphA3 is a component of the Eph/ephrin tyrosine kinase system, which participates in vasculature development. This receptor/ligand system is associated with various signaling pathways related to cell growth and viability, cytoskeletal organization, cell migration, and anti-apoptosis. Accumulated evidence suggests that aberrant regulation of EphA3 and its genetic alterations are implicated in the development and progression of various cancers. However, despite a high incidence of EphA3 over-expression, no such investigation has been performed in hepatocellular carcinoma. Thus, we investigated genetic alterations of the EphA3 gene in 73 cases of hepatocellular carcinoma by single-strand conformational polymorphism and sequencing. One novel D219V missense mutation was found in the extracellular domain of EphA3, and two genetic alterations in the intracellular sterile-alpha-motif (SAM) domain of EphA3 appeared to be polymorphisms. Although the functional assessments of this mutant are incomplete, it is believed that this novel EphA3 mutation may contribute to the development of hepatocellular carcinoma.

Mutational analysis of JAK1 gene in human hepatocellular carcinoma.

UNLABELLED: The Janus kinase 1 (JAK1) gene encodes a cytoplasmic tyrosine kinase that is noncovalently associated with a variety of cytokine receptors and plays a nonredundant role in cell proliferation, survival, and differentiation. The mutated forms of JAK1 often altered the activation of JAK1 and then changed the activation of JAK1/STAT pathways, and this may contribute to cancer development and progression. Thus, to investigate whether genetic mutations of JAK1 gene are associated in hepatocellular carcinoma (HCC) progression, we analyzed genetic alterations of JAK1 gene in 84 human HCCs by single-strand conformational polymorphism (SSCP) and direct sequencing. Of 24 exons of JAK1 gene, 12 exons were previously reported to have mutations, we searched genetic alteration of JAK1 in these exons. Overall, one missense mutation (1.2%) was found. In addition, 12 cases (14%) were found to have single nucleotide polymorphism (14%) in exon 14. Taken together, we found one novel missense mutation of JAK1 gene in hepatocellular carcinomas with some polymorphisms. Although the functional assessment of this novel mutant remains to be completed, JAK1 mutation may contribute to the tumor development in liver cancer. KEYWORDS: JAK1 gene, hepatocellular carcinoma, mutation.

Mutational analysis of oncogenic AKT E17K mutation in common solid cancers and acute leukaemias.

Mounting evidence indicates that alterations of AKT signalling play important roles in cancer development. An earlier study discovered an oncogenic AKT1 gene mutation (AKT1 E17K) in breast, colorectal and ovarian cancers. The aim of this study was to see whether the AKT1 E17K mutation is common in breast, colorectal, lung, gastric and hepatocellular carcinomas and acute leukaemias. We analysed the presence of the AKT1 E17K mutation in 731 cancer tissues by a single-strand conformation polymorphism assay. In addition, we analysed the corresponding sequences of AKT1 E17K in AKT2 and AKT3 genes. Overall, we detected the four AKT1 E17K mutations in the breast cancers (4/93; 4.3%), but none in other cancers. There was no AKT2 or AKT3 mutation in the cancers. This study demonstrated that the AKT1 E17K mutation occurs in breast cancers at a low frequency, and that it is rare in other common cancers, including colorectal, lung, gastric and hepatocellular carcinomas and acute leukaemias. Despite the confirmed oncogenic function of the AKT1 E17K, the rare incidences of the mutation suggest that it may not play a crucial role in the development of the most common types of human cancers.

KLF6 IVS1 -27G/A polymorphism with susceptibility to gastric cancers in Korean.

KLF6 is a key cell cycle regulator that is downregulated in several kinds of human cancers, including gastric cancer. The IVS1 -27G/A polymorphism of KLF6 has been investigated, which can influence susceptibility to gastric cancer and disease outcome. In order to investigate whether the IVS1 -27G/A polymorphism of KLF6 is associated with individual susceptibility to gastric cancer in Korea, the frequency of the polymorphism was examined in 264 gastric cancer patients and 299 healthy controls. Single nucleotide polymorphism (SNP) analysis was performed by amplifying intron 1 of KLF6 and sequencing the products. The frequencies of genotypes: G/G, G/A and A/A were 91.7% (242/264), 5.7% (15/264) and 2.6%, respectively, in gastric cancer cases and 91.9%, 7.0% and 1.1%, respectively, in healthy controls. Genotype frequencies in Korean population were very similar to those of Caucasian population. Interestingly, the male gastric cancer patients showed a significantly higher proportion of the G allele (Chi-Square test, P=0.005) compared to female gastric cancer patients. However, the polymorphism was statistically not associated with increased risk of gastric cancer in Korea. When stratified by histological subtype of gastric cancer, the risk was also not statistically significant. Thus, our results suggested that the IVS1 -27G/A polymorphism of KLF6 is not associated with an increased risk for gastric cancer in Korean population.

Chk1 frameshift mutation in sporadic and hereditary non-polyposis colorectal cancers with microsatellite instability.

AIM: Protein kinase Chk1 (hChk1) is essential in human cells for cell cycle arrest in response to DNA damage, and has been shown to play an important role in the G2/M checkpoint. The BRAF mutations have been suggested to be linked with defective mismatch repair in colorectal cancers. The aim of this study was to investigate whether a frameshift mutation within the Chk1 gene contribute to the development or progression of eastern sporadic and hereditary non-polyposis colorectal cancer (HNPCC) with microsatellite instability (MSI). METHODS: We analyzed MSI using the 6 microsatellite markers and a frameshift mutation in the BRAF gene and in poly(A)9 within the Chk1 gene in 51 sporadic colorectal cancer and 14 HNPCC specimens. RESULTS: Eleven of the 51 sporadic colorectal cancers and all of the 14 HNPCCs were MSI-positive. Chk1 frameshift mutations were observed in 2 and 3 sporadic colon cancers and HNPCC, respectively, whereas no BRAF mutations were detected in these samples. Interestingly, all cases with the Chk1 frameshift mutation had high-frequency MSI. CONCLUSION: These results suggest that the Chk1 gene is a target of genomic instability in MSI-positive colorectal cancers and that the Chk1 framshift mutations might be involved in colorectal tumourigenesis through a defect in response to DNA damage in a subset of sporadic colorectal cancers and HNPCCs.

Association of cyclin D1 G870A polymorphism with susceptibility to gastric cancers in Korean male patients.

Cyclin D1 is a key cell cycle regulator that is upregulated in gastric cancer. The common G870A polymorphism of cyclin D1 which can influence cancer susceptibility and disease outcome has been the most frequently investigated. The specific aim of this study is to investigate whether the G870A polymorphism of cyclin D1 was associated with individual susceptibility to gastric cancer in Korea. The frequency of the polymorphism was examined in 253 gastric cancer patients and 442 healthy controls. Polymorphism analysis was performed by amplifying exon 4 of cyclin D1 and sequencing the products. The frequencies of genotypes: G/G, G/A and A/A were 28.1% (71/253), 49.4% (125/253) and 22.5% (57/253), respectively, in gastric cancer cases, and 23.1%, 51.1% and 25.8%, respectively, in healthy controls. Statistically, the polymorphism was not associated with increased risk of gastric cancer. When stratified by histological subtype of gastric cancer, the risk was also not statistically significant. However, the male gastric cancer patients showed a significantly higher proportion of the homozygous G/G genotype and the G allele (Chi-Square test, P = 0.0242 & P = 0.0307) compared to males in the control group. Thus, our findings suggested that the G870A polymorphism of cyclin D1 was not associated with an increased risk for gastric cancer in this population, however, it may contribute to susceptibility to gastric cancer in men.

Immunohistochemical analysis of pro-apoptotic PUMA protein and mutational analysis of PUMA gene in gastric carcinomas.

BACKGROUND: Mounting evidence indicates that alteration of apoptosis is involved in the mechanisms of cancer development. PUMA, a pro-apoptotic member of Bcl-2 family, mediates p53-dependent and -independent apoptosis. AIM: The aim of this study was to explore whether alteration of PUMA protein expression is a characteristic of human gastric carcinomas. PATIENTS AND METHODS: We analysed expression of PUMA protein in 60 gastric adenocarcinomas by immunohistochemistry. Also, we examined PUMA gene mutation in the same tissues by a single-strand conformation polymorphism. RESULTS: PUMA protein expression was detected in 44 cases (73%) of the 60 gastric carcinomas, whereas it was not detected in normal gastric mucosal epithelial cells. The mutational analysis revealed no PUMA mutation in the gastric carcinomas. CONCLUSIONS: Our data suggest that PUMA mutation is not a direct target of inactivation in gastric tumourigenesis. Also, increased expression of PUMA in malignant gastric epithelial cells compared with normal mucosal epithelial cells suggested that PUMA expression may play a role in gastric tumourigenesis.

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.

Expressional analysis of anti-apoptotic phospho-BAD protein and mutational analysis of pro-apoptotic BAD gene in hepatocellular carcinomas.

BACKGROUND: It has become clear that, together with proliferation, deregulation of apoptosis plays a pivotal role in tumourigenesis. BAD is a pro-apoptotic Bcl-2 family protein and regulates the intrinsic apoptosis pathway. Phosphorylation of BAD inhibits the apoptosis function of BAD. AIMS: To investigate whether alteration of the phospho-BAD protein and somatic mutation of BAD gene are characteristics of human hepatocellular carcinoma. PATIENTS AND METHODS: We analysed the expression of phospho-BAD in 20 hepatocellular carcinomas by immunohistochemistry. Also, we analysed the BAD gene for the detection of somatic mutations by a single-strand conformation polymorphism assay in 69 hepatocellular carcinomas. RESULTS: Phospho-BAD expression in the non-tumour hepatocytes was seen in all of the hepatocellular carcinomas, while the expression in the cancer cells was observed in 15% (3 of the 20) of the hepatocellular carcinomas. There was no somatic mutation of BAD Bcl-2 homology 3 (BH3) domain in the 69 hepatocellular carcinomas. CONCLUSIONS: The data showed that loss of phospho-BAD expression, but not BAD gene mutation, is a feature of hepatocellular carcinomas. The decreased expression of phospho-BAD in the hepatocellular carcinoma cells compared to the non-tumour hepatocytes suggests that loss of phospho-BAD expression may play a role in hepatocellular tumourigenesis.

The JAK2 V617F mutation in de novo acute myelogenous leukemias.

A missense somatic mutation in JAK2 gene (JAK2 V617F) has recently been reported in chronic myeloproliferative disorders, including polycythemia vera, essential thrombocythemia and myelofibrosis with myeloid metaplasia, strongly suggesting its role in the pathogenesis of myeloid disorders. As activation of JAK2 signaling is occurred in other malignancies as well, we have analysed 558 tissues from common human cancers, including colon, breast and lung carcinomas, and 143 acute adulthood leukemias by polymerase chain reaction -- single strand conformation polymorphism analysis. We found three JAK2 mutations in the 113 acute myelogenous leukemias (AMLs) (2.7%), but none in other cancers. The mutations consisted of two V617F mutations and one K607N mutation. None of the AML patients with the JAK2 V617F mutation had a history of previous hematologic disorders. This is the first report on the JAK2 gene mutation in AML, and the data indicated that the JAK2 gene mutation may not only contribute to the development of chronic myeloid disorders, but also to some AMLs.

Genetic analysis of the LKB1/STK11 gene in hepatocellular carcinomas.

The tumour suppressor gene, LKB1/STK11, has been mapped to chromosome 19p13, a region showing frequent allelic loss in various human cancers, including hepatocellular carcinoma (HCC). Additionally, LKB1 physically associates with p53 and regulates p53-dependent apoptotic pathways. To investigate whether genetic alterations of LKB1 could be involved in the tumorigenesis of HCC, we analysed the genetic alterations of the LKB1 and p53 genes in seven dysplastic nodules and 80 HCCs. We found one LKB1 missense mutation, CCG-->CTG (Pro-->Leu) at codon 281 within the kinase domain. We also found allelic loss in six of 27 (22%) informative HCC cases and all of them were HBV-positive cases. In addition, we detected seven missense, one nonsense and one silent mutations (nine of 80, 11%) of p53 in HCCs only. These results suggest that genetic alterations of the LKB1 or p53 genes may play an important role in tumour development or progression of a sub-set of HCCs, and may also provide alternative mechanisms to protect the HCC cell from p53-dependent apoptosis.