Kyoko Iwao

Association of centrosomal kinase STK15/BTAK mrna expression with chromosomal instability in human breast cancers

Over‐expression of a centrosomal serine/threonine kinase, STK15/BTAK, induces centrosome amplification, which results in chromosomal instability (CIN) in cell culture. In the present study, we investigated the correlation of STK15/BTAK mRNA expression with CIN and various clinicopathological factors in human breast cancer. STK15/BTAK mRNA levels were quantified by real‐time PCR, and CIN values were determined by FISH analysis of chromosomes 1, 11 and 17 using centromeric probes. STK15/BTAK mRNA levels (0.310 ± 0.413, mean ± SD, n = 47) in breast cancers were significantly (p < 0.01) higher than those in normal breast tissues (0.044 ± 0.029, n = 9). Furthermore, breast cancers were divided into 3 groups (low, intermediate and high) according to STK15/BTAK mRNA expression levels. CIN values of the low‐expression group (27.9 ± 12.6%, n = 18) were significantly (p < 0.01) higher than those of normal breast tissues (9.2 ± 2.6%, n = 6), and those of the high‐expression group (38.0 ± 12.7%, n = 14) were significantly (p < 0.05) higher than those of the low‐expression group. STK15/BTAK mRNA expression showed a significant (p < 0.05) correlation with high histological grade and negativity of estrogen and progesterone receptors. Our results demonstrate that STK15/BTAK mRNA is over‐expressed in the majority of breast cancers and its over‐expression is significantly associated with CIN, implicating STK15/BTAK in carcinogenesis through induction of CIN. STK15/BTAK mRNA levels might be useful as an indicator of poor prognosis and resistance to endocrine therapy.

Quantitative analysis of estrogen receptor‐β mRNA and its variants in human breast cancers

We have carried out a quantitative analysis of ER‐α and ER‐β mRNA expression in normal (n = 11) and breast cancer (n = 112) tissues using a real‐time (Taq‐Man) PCR assay. Expression of ER‐β mRNA variants has also been studied by triple‐primer PCR assay. ER‐α mRNA levels in normal breast tissues were significantly (p < 0.01) lower than those in ER‐positive breast cancers but not significantly different from those in ER‐negative breast cancers. However, ER‐β mRNA levels in normal breast tissues were significantly (p < 0.01) higher than those in ER‐positive and ER‐negative breast cancers. Proportions of ER‐β1 and ER‐β2 mRNA expression among total ER‐β mRNA expression were significantly higher and those of ER‐β5 and ER‐β5` mRNA were significantly lower in normal breast tissues than in ER‐positive and ER‐negative breast cancers. ER‐β mRNA levels and proportions of ER‐β mRNA variants did not show any significant correlation with age, tumor size, lymph node status and histological grade. Our results demonstrate that ER‐α mRNA is up‐regulated and ER‐β mRNA is down‐regulated during carcinogenesis of breast cancers. Changes in proportions of ER‐β mRNA variants are also implicated in this process. Int. J. Cancer 88:733–736, 2000.

Isolation of a novel human lung‐specific gene, LUNX, a potential molecular marker for detection of micrometastasis in non‐small‐cell lung cancer

We have isolated a novel human lung‐specific gene, LUNX (lung‐specific X protein), by differential‐display mRNA analysis. The full‐length cDNA contained 1,015 nucleotides including an open reading frame of 768 nucleotides encoding 256 amino acids. We localized the gene to chromosomal region 20p11.1‐q12 by radiation hybrid mapping. Using an RT‐PCR assay specific for LUNX mRNA, 35 non‐small‐cell lung‐cancer (NSCLC) tumors and 0 of 16 normal lymph nodes were positive. Furthermore, LUNX mRNA expression was enhanced in 26 (84%) of 31 NSCLC tumors vs. corresponding cancer‐free lung tissues by semi‐quantitative analyses with multiplex RT‐PCR. We assessed the possibility of LUNX mRNA as a molecular marker for detection of micrometastasis in dissected lymph nodes obtained from 20 patients with NSCLC tumors. LUNX mRNA was detected in 16 (80%) of 20 histologically positive lymph nodes and 21 (25%) of 84 histologically negative lymph nodes. Comparative analyses of the conventional histological examination and the RT‐PCR detection assay for LUNX mRNA showed that the detection rate of metastases in lymph nodes by the RT‐PCR assay was higher in 12 and consistent in 6 of the total 20 NSCLC patients. We demonstrate that the LUNX RT‐PCR assay is a potential diagnostic method for detection of micrometastases in lymph nodes of NSCLC patients.

Quantitative analysis of estrogen receptor‐α and ‐β messenger RNA expression in breast carcinoma by real‐time polymerase chain reaction

Estrogen action is mediated not only through a classic estrogen receptor (ER) (ER‐α) but also through a second ER (ER‐β) that has a structure and function similar to ER‐α. A correlation between ER‐β mRNA expression with ER and progesterone receptor (PR) protein levels as well as prognostic factors remains to be established in breast carcinoma.

Breast cancer risk associated with polymorphism in CYP19 in Japanese women

Screening of the entire coding and major promoter regions of the CYP19 gene identified two novel polymorphisms at codon 39 (Trp to Arg) and codon 408 (silent) in addition to those reported previously at codon 264 (Arg to Cys) and intron 4 [tetranucleotide (TTTA) simple tandem repeat]. A case‐control study was conducted in order to see whether or not these polymorphisms were associated with breast cancer risk in Japanese women. Homozygous and heterozygous carriers of the variant allele Arg at codon 39 showed a significantly decreased risk of breast cancer (OR=0.39, 95%C.I.=0.17–0.89). On the other hand, homozygous carriers of the allele with 10 or more TTTA repeats at intron 4 showed a trend toward an increase (OR=1.80, 95%C.I.=0.97–3.36) in breast cancer risk. Other polymorphisms were found not to be associated with breast cancer risk. These results suggest that the CYP19 polymorphisms at exon 39 and intron 4 would be useful for selecting Japanese women at a high risk of breast cancer. Int. J. Cancer 89:325–328, 2000.

Frequent β-Catenin Abnormalities in Bone and Soft-tissue Tumors

We have screened mutations of the β‐catenin gene by using the polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) method in 62 malignant bone and soft‐tissue tumors, including malignant fibrous histiocytomas (MFHs), osteosarcomas, synovial sarcomas, liposarcomas, malignant schwannomas, and other types of tumors, as well as 11 benign tumors. β‐Catenin‐activating missense mutations were found in two malignant tumors. One found in MFH occurred at codon 45 and caused an amino acid substitution from serine (one of the GSK3β‐targeted phosphorylation sites) to phenylalanine. The other, detected in synovial sarcoma at codon 32, resulted in an amino acid change from aspartic acid (located adjacent to the phosphorylation target, serine, encoded by codon 33) to tyrosine. Furthermore, we found accumulation of β‐catenin by western‐blotting analysis in 12 of 19 malignant tumors in which we found no mutation involving exon 3. Our results suggested the possible involvement of β‐catenin activation, by β‐catenin gene mutation or alteration of other factor(s), in the formation and/or progression of various types of bone and soft‐tissue tumors.

Genetic polymorphism in CYP17 and breast cancer risk in Japanese women.

A case-control study was conducted to investigate the association of two genetic polymorphisms (1931T/C and 1951G/A) in the promoter region of the CYP17 gene with breast cancer risk in Japanese women. No significant association was observed between CYP17 polymorphism(1951G/A) and breast cancer risk (odds ratio (OR) = 1.71, 95% confidence interval (CI): 0.28-1.84). In contrast, a significant increase in breast cancer risk (OR= 1.82. 95% CI: 1.07-3.12) was observed in CYP17(1931C/C) homozygotes compared with CYP17(1931T/C) heterozygotes and CYP17(1931T/T) homozygotes when women aged > or = 55 years were considered, but such a significant increase was not observed when women aged < or = 54 years were considered (OR = 0.96, 95% CI: 0.56-1.63). These results suggest that CYP17 polymorphism(1931T/C) would be useful in the selection of Japanese women at a high risk for developing breast cancer at the age of > or = 55 years.

Mammaglobin B gene as a novel marker for lymph node micrometastasis in patients with abdominal cancers.

Mammaglobin B is a recently-isolated gene speculated to belong to the uteroglobin gene family and is overexpressed in primary breast cancers. We investigated mammaglobin B mRNA expression in various cancers of the digestive system. Given the absence of mammaglobin B expression in normal lymph nodes, we also assessed the usefulness of mammaglobin B as a marker for lymph node micrometastases in cancer patients. Mammaglobin B gene transcripts were frequently detected by reverse transcriptase-polymerase chain reaction (RT-PCR) assay in primary tumors of the esophagus (2/3), stomach (7/7), colon (15/15), pancreas (4/6), common bile duct (6/6), cholangioma (2/2) and gall bladder (1/1). Mammaglobin B overexpression was observed in three of 15 cases (20%) of colon cancer, suggesting its possible contribution to colon carcinogenesis. Down-regulated mammaglobin B expression was observed in hepatoma cells in comparison with corresponding non-cancerous livers (3/3). RT-PCR assay of mammaglobin B detected 14 of 15 histologically positive lymph nodes from patients with gastric cancer, colon cancer and cholangioma. Seven of 32 (22%), three of nine (33%), and three of seven (43%) histologically negative nodes from patients with gastric, colon and cholangiocellular carcinoma, respectively, were found to express mammaglobin B mRNA. Our results showed that expression of mammaglobin B was frequently detected in cancers originating in digestive organs, especially adenocarcinomas, and that mammaglobin B gene detected by RT-PCR may be a potentially useful molecular marker for lymph node micrometastases of various digestive organ cancers.

Quantitative Analysis of Estrogen Receptor-α and -β Messenger RNA Expression in Normal and Malignant Thyroid Tissues by Real-Time Polymerase Chain Reaction

Objectives: We have conducted a quantitative analysis of estrogen receptor-α (ER-α) and -β (ER-β) mRNA expression in normal thyroid and tumor tissues. Methods: Normal thyroid tissues (n = 10) and tumor tissues [(follicular adenoma (n = 14), follicular carcinoma (n = 8), papillary carcinoma (n = 14), anaplastic carcinoma (n = 3) and medullary carcinoma (n = 6)] were obtained at surgery from 45 female patients. ER-α and ER-β mRNA expression has been studied by a quantitative polymerase chain reaction. Results: ER-α mRNA levels in the normal thyroid were not significantly different from those in follicular adenomas, papillary carcinomas and medullary carcinomas but were marginally (p = 0.08) higher than those in follicular and anaplastic carcinomas. ER-β mRNA levels in the normal thyroid tissues were not significantly different from those in any other tumor tissues. ER-β to ER-α mRNA ratios were significantly (p < 0.05) higher in the normal thyroid tissues than in follicular adenomas. Proportions of ER-β mRNA variants (ER-β 1, 2, 5, and 5′) did not significantly differ among the normal and tumor tissues. Conclusions: A downregulation of ER-α mRNA in follicular and anaplastic carcinomas seems to suggest that estrogens are unlikely to play an important role in the carcinogenesis and progression of these carcinomas. On the other hand, a significant decrease in ER-β to ER-α mRNA ratios in follicular adenomas suggests a possible involvement of estrogens in the pathogenesis of this disease since the same phenomenon has been reported on estrogen-dependent breast cancers.

Molecular and biological analysis of carcinoma of the small intestine: β -catenin gene mutation by interstitial deletion involving exon 3 and replication error phenotype

The genetic mechanisms of carcinomas of the small intestine are not well understood. We report the results of analysis of genetic alterations in a case of small intestinal carcinoma. A tumor in the terminal ileum was resected in a 59-yr-old woman. Histologically, the tumor was classified as well-differentiated adenocarcinoma. We screened for genetic alterations in adenomatous polyposis coli (APC), β-catenin, K-ras, and p53 genes, as well as microsatellite instability, which are known to be involved in colorectal tumorigenesis. The tumor exhibited somatic interstitial deletion of 425-bp, which included the entire exon 3 in β-catenin gene. Immunohistochemical staining confirmed accumulation of aberrant β-catenin protein in the cytoplasm and nuclei of the malignant tissue. Furthermore, a frameshift mutation in the transforming growth factor β receptor type II gene with replication error phenotype was detected in the tumor DNA. In contrast, no genetic alterations were found in the APC, K-ras, and p53 genes. Our results suggested that both β-catenin gene mutation and replication error phenotype might contribute to carcinogenesis of the small intestinal tumor in our case. This is the first report that activation of β-catenin gene by somatic gene mutation is involved in the development of carcinoma of the small intestine.