Takeshi Iwaya

Downregulation of miR-138 is associated with overexpression of human telomerase reverse transcriptase protein in human anaplastic thyroid carcinoma cell lines

Alterations of several microRNA (miRNA) have been linked to cancer development and its biology. To search for unique miRNA that might play a role in the development of anaplastic thyroid carcinoma (ATC), we examined the expression of multiple miRNA and their functional effects on target genes in human thyroid carcinoma cell lines. We quantitatively evaluated the expression of multiple miRNA in 10 ATC and five papillary thyroid carcinoma (PTC) cell lines, as well as primary tumors from 11 thyroid carcinoma patients (three ATC and eight PTC), using the stem‐loop‐mediated reverse transcription real‐time polymerase chain reaction method. We also examined the target gene specificity of unique miRNA that showed differences in expression between ATC and PTC cell lines. One miRNA, miR‐138, was significantly downregulated in ATC cell lines in comparison with PTC (P < 0.01). Eleven miRNA (including miR‐138) potentially targeting the human telomerase reverse transcriptase (hTERT) gene were totally downregulated in both ATC and PTC cell lines in comparison with normal thyroid tissues. A tendency for an inverse correlation between miR‐138 and hTERT protein expression was observed in the thyroid cancer cell lines, although this failed to reach significance (r = –0.392, P = 0.148). We demonstrated that overexpression of miR‐138 induced a reduction in hTERT protein expression, and confirmed target specificity between miR‐138 and the hTERT 3′‐untranslated region by luciferase reporter assay. These results suggest that loss of miR‐138 expression may partially contribute to the gain of hTERT protein expression in ATC, and that further multiple miRNA targeting hTERT mRNA might be involved in the development of thyroid carcinoma. (Cancer Sci 2008; 99: 280–286)

Clinical significance of the expression of long non-coding RNA HOTAIR in primary hepatocellular carcinoma.

The functions of many long non-coding RNAs (ncRNAs) in human cancers have not yet been elucidated. The long ncRNA HOTAIR is expressed from the developmental HOXC locus located on chromosome 12q13.13. Previous reports have demonstrated that HOTAIR associates with chromatin modifications in cooperation with the Polycomb complex PRC2, and promotes breast and colorectal cancer metastasis. In this study, we examined the clinical significance of HOTAIR expression in patients with hepatocellular carcinoma (HCC). HOTAIR expression was detected in primary HCCs in 13 out of 64 patients. Patients with HOTAIR expression had significantly poorer prognoses and a larger primary tumor size than those without HOTAIR expression, similar to studies in breast and colorectal cancers. Moreover, introduction of human HOTAIR into liver cancer cells revealed that HOTAIR promoted more rapid proliferation compared to control cells. Thus, although the clinical significance of HOTAIR expression in HCC may not be as pronounced as that in breast and colorectal cancers, the current study demonstrates that HOTAIR expression is associated with HCC progression, warranting further studies.

Plastin3 Is a Novel Marker for Circulating Tumor Cells Undergoing the Epithelial–Mesenchymal Transition and Is Associated with Colorectal Cancer Prognosis

Circulating tumor cells (CTC) in blood have attracted attention both as potential seeds for metastasis and as biomarkers. However, most CTC detection systems might miss epithelial-mesenchymal transition (EMT)-induced metastatic cells because detection is based on epithelial markers. First, to discover novel markers capable of detecting CTCs in which EMT has not been repressed, microarray analysis of 132 colorectal cancers (CRC) from Japanese patients was conducted, and 2,969 genes were detected that were overexpressed relative to normal colon mucosa. From the detected genes, we selected those that were overexpressed CRC with distant metastasis. Then, we analyzed the CRC metastasis-specific genes (n = 22) to determine whether they were expressed in normal circulation. As a result, PLS3 was discovered as a CTC marker that was expressed in metastatic CRC cells but not in normal circulation. Using fluorescent immunocytochemistry, we validated that PLS3 was expressed in EMT-induced CTC in peripheral blood from patients with CRC with distant metastasis. PLS3-expressing cells were detected in the peripheral blood of approximately one-third of an independent set of 711 Japanese patients with CRC. Multivariate analysis showed that PLS3-positive CTC was independently associated with prognosis in the training set (n = 381) and the validation set [n = 330; HR = 2.17; 95% confidence interval (CI) = 1.38-3.40 and HR = 3.92; 95% CI = 2.27-6.85]. The association between PLS3-positive CTC and prognosis was particularly strong in patients with Dukes B (HR = 4.07; 95% CI = 1.50-11.57) and Dukes C (HR = 2.57; 95% CI = 1.42-4.63). PLS3 is a novel marker for metastatic CRC cells, and it possesses significant prognostic value.

Mutations in mitochondrial control region DNA in gastric tumours of Japanese patients.

The non-coding control region of mitochondrial DNA (mtDNA), containing the hypervariable regions HV1 and HV2 and the D-loop region, was screened for mutations in 45 gastric tumours (15 tumours each of adenoma, differentiated adenocarcinoma and undifferentiated carcinoma). We found mutations in two of the 45 tumours (4%); a 1 bp A deletion at nucleotide position 248 in a differentiated adenocarcinoma and a G to A transition at nucleotide position 16,129 in an adenoma. We also observed 10 polymorphisms, four of which were not previously recorded. Both mtDNA mutations were present in replication error negative (RER-) tumours. Short mono- or dinucleotide repeats in the control region, such as (C)7, (A)5 or (CA)5, were not altered regardless of nuclear genetic instability. In summary, mtDNA is mutated in a subset of benign and malignant gastric tumours, but, disruption of the mtDNA repair system appears not to be significantly involved in gastric tumours of Japanese patients.

Inactivation of the E-Cadherin Gene in Primary Gastric Carcinomas and Gastric Carcinoma Cell Lines

We investigated the E (epithelial)‐cadherin gene for mutations and loss of heterozygosity (LOH) in 24 primary gastric carcinomas (12 differentiated and 12 undifferentiated types, including 3 signet‐ring cell carcinomas), as well as 4 gastric carcinoma cell lines of the undifferentiated type (MKN‐45, GCIY, HGC‐27 and GT3TKB). We utilized PCR‐SSCP and RT‐PCR followed hy direct sequencing to detect gene mutations and skipped exons, and RT‐PCR‐SSCP to examine LOH. In primary carcinomas, gene mutations or skipped exons, were detected in 4 of 9 (44%) undifferentiated carcinomas of the scattered type, including 2 signet‐ring cell carcinomas, and in none of the 3 undifferentiated carcinomas of the adherent type and 12 differentiated carcinomas. Demonstrated mutations of the E‐cadherin gene included an 18 bp deletion (codon 418‐423) and a 3 bp deletion (codon 400, calcium‐binding domain), both located in exon 9. Skipping of exon 9 with a 1 bp insertion at codon 337, and skipping of exon 8 with a 1 bp deletion at codon 336, also were detected. LOH was confirmed in all of the carcinomas in which gene mutations or skipped exons (3/3 informative cases) were demonstrated. The MKN‐45 cell line exhibited an 18 bp deletion at the exon 6‐intron 6 boundary with loss of the wild‐type allele, and 2 of the remaining 3 cell lines (HGC‐27 and GT3TKB) had lost expression without detectable structural alteration of the E‐cadherin gene. These data provide support for classic two‐hit inactivation of the E‐cadherin gene in a high percentage of undifferentiated carcinomas of the scattered type.

Commonly deleted regions on the long arm of chromosome 21 in differentiated adenocarcinoma of the stomach

During an allelotype analysis of differentiated adenocarcinoma of the stomach, we observed frequent loss of heterozygosity (LOH) on several chromosomes including the long arm of chromosome 21 (21q). Therefore, we analyzed DNA isolated from 45 tumors for LOH at 10 loci on 21q by using polymorphic microsatellite markers. In 20 (44%) of 45 tumors, we detected LOH at single or multiple loci on 21q. Deletion mapping of these 20 tumors revealed two separate commonly deleted regions. Our findings suggest that 21q contains at least two potential tumor suppressor genes which play crucial roles in the development of differentiated adenocarcinoma of the stomach. Genes Chromosom. Cancer 18:318–321, 1997.

Mutations in the human homologue of the Drosophila patched gene in esophageal squamous cell carcinoma

The human homologue (PTCH) of the Drosophila segment polarity gene patchedhas recently been identified as a tumor‐suppressor gene for nevoid basal cell carcinoma syndrome and for sporadic basal cell carcinomas of the skin. We analyzed 30 esophageal squamous cell carcinomas (ESCC) for intrageneic mutations of the PTCH gene by polymerase chain reaction–single‐strand conformation polymorphism analysis followed by DNA sequencing. We identified two somatic PTCH mutations (7%) in 30 ESCC. These were a nonsense mutation (CAG to TAG at codon 361) in exon 8 and a missense mutation (CAG to CTG, Gln to Leu at codon 816) in exon 14. These tumors exhibited loss of heterozygosity at the polymorphic site of the PTCH gene. These results indicate that inactivation of the PTCH gene via a two‐hit mechanism occurs in a subset of ESCC. Genes Chromosomes Cancer 21:276–279, 1998.

Tylosis esophageal cancer locus on chromosome 17q25.1 is commonly deleted in sporadic human esophageal cancer

BACKGROUND & AIMS Tumor-suppressor genes found in inherited cancer predisposition syndromes are also responsible for sporadic cancers of the same type. Recently, the tylosis oesophageal cancer (TOC) gene locus has been mapped to 17q25 by linkage analyses of pedigrees with focal nonepidermolytic palmoplantar keratoderma associated with a high risk of esophageal cancer development. The aim of this study was to clarify whether the TOC locus is affected in sporadic esophageal cancers. METHODS We investigated loss of heterozygosity (LOH) on 17q in 58 sporadic esophageal squamous cell carcinomas (ESCs) using 20 microsatellite markers focusing on the TOC locus. RESULTS LOH on 17q was observed in 37 of 52 (71%) informative cases at one or more loci, 80% (33/37) of which included the TOC locus. The smallest common deleted region was at D17S1839 within the TOC locus. CONCLUSIONS The constructed deletion map revealed that the TOC locus is commonly deleted in sporadic ESCs, suggesting that a tumor-suppressor gene responsible for ESC is contained within this locus.

Esophageal carcinosarcoma: A genetic analysis

Carcinosarcoma of the esophagus is a rare malignant neoplasm that consists of both carcinomatous and sarcomatous elements. The histogenesis of the sarcomatous component is generally considered to result from metaplasia of carcinomatous cells toward mesenchymal differentiation. True carcinosarcoma, characterized as a collision between a carcinoma and a sarcoma, is extremely rare. We describe a patient with primary achalasia who developed a true carcinosarcoma of the esophagus in which clonal differences between carcinomatous and sarcomatous elements were genetically and immunohistochemically demonstrated. A polypoid tumor located in the middle third of the esophagus developed in a 51-year-old man with longstanding achalasia. The tumor was predominantly composed of spindle-shaped sarcomatous cells. Squamous cell carcinoma in situ and islands of well-differentiated squamous cell carcinoma in the sarcomatous element were histologically observed. The sarcomatous element was immunoreactive for both mesenchymal and myoid markers. The carcinomatous component expressed type I and type II cytokeratins as well as epithelial membrane antigen. Analysis for chromosomal loss of heterozygosity performed in multiple microdissected samples of each sarcomatous and carcinomatous element revealed distinct genetic clonalities. These differences in immunohistochemical and genetic clonalities suggest that the tumor composed of squamous cell carcinoma and leiomyosarcoma originated separately from epithelial and mesenchymal precursors.

Integrated Multiregional Analysis Proposing a New Model of Colorectal Cancer Evolution.

Understanding intratumor heterogeneity is clinically important because it could cause therapeutic failure by fostering evolutionary adaptation. To this end, we profiled the genome and epigenome in multiple regions within each of nine colorectal tumors. Extensive intertumor heterogeneity is observed, from which we inferred the evolutionary history of the tumors. First, clonally shared alterations appeared, in which C>T transitions at CpG site and CpG island hypermethylation were relatively enriched. Correlation between mutation counts and patients’ ages suggests that the early-acquired alterations resulted from aging. In the late phase, a parental clone was branched into numerous subclones. Known driver alterations were observed frequently in the early-acquired alterations, but rarely in the late-acquired alterations. Consistently, our computational simulation of the branching evolution suggests that extensive intratumor heterogeneity could be generated by neutral evolution. Collectively, we propose a new model of colorectal cancer evolution, which is useful for understanding and confronting this heterogeneous disease.