Shingo Akimoto

Expression of E-Cadherin, α-Catenin, β-Catenin and Plakoglobin in Esophageal Carcinomas and Its Prognostic Significance

It has been suggested that inactivation of the cadherin-mediated cell-cell adhesion system plays a role in the initial steps of cancer invasion and metastasis. Expression of E-cadherin and its intracy

A faithful method for PCR-mediated global mRNA amplification and its integration into microarray analysis on laser-captured cells.

Quantitative and qualitative analyses of mRNAs from a small number of cells are extremely important for studies on gene expression in various physiological and pathological conditions in multicellular organisms. We present here an effective method for high-fidelity global mRNA amplification for in vivo gene expression profiling of as few as 100 cells obtained by laser-captured microdissection (LCM). This method, called TALPAT, is based on T7 RNA polymerase-mediated transcription, adaptor ligation, and PCR amplification followed by T7-transcription. More than 80% of genes were commonly identified as a more than 3-fold changed gene among three gastric cancer cell lines using cRNA amplified by both TALPAT and the ordinary in vitro T7-transcription. The reproducibility of TALPAT was validated by microarray analysis on 100 breast cancer cells obtained by LCM. For the application of the LCM-TALPAT method, we successfully obtained expression profiles of gastric cancer cells and the mesenchymal cells, enabling us to understand in vivo cell-to-cell cross-talk in the microenvironment.

Frequent Loss of α Catenin Expression in Scirrhous Carcinomas with Scattered Cell Growth

To investigate the mechanisms of disruption of cell‐cell contact in scirrhous carcinoma cells, the expression of both E‐cadherin and α catenin, which is an intracellular cadherin‐binding molecule, were determined in scirrhous‐type adenocarcinomas of the stomach and breast using immunohistochemical and immunoblotting techniques. The losses of E‐cadherin expression in gastric and breast scirrhous adenocarcinomas were 18.1% and 0%, respectively, and those of α catenin expression were 54.6% and 75%, respectively. Frequent loss of α catenin expression occurred in scirrhous carcinomas with scattered cell growth in the stomach and the breast and showed no organ specificity. In addition, all the infiltrating lobular carcinomas, which also infiltrate the stroma as single cells, showed no E‐cadherin or α catenin expression. These findings suggest that down‐regulation of either α catenin or E‐cadherin plays a critical role in the disruption of cell adhesion in carcinomas with scattered cell growth.

Cytoplasmic beta-catenin accumulation as a predictor of hematogenous metastasis in human colorectal cancer.

The membranous, cytoplasmic and nuclear levels of beta-catenin were evaluated immunohistochemically in archival tissue specimens from 96 Japanese patients with primary colorectal carcinoma who had undergone surgery. The relationships between beta-catenin and clinicopathological variables were analyzed statistically. Reduced beta-catenin immunoreactivity in the cell membranes of cancer cells was found in 70% of the tumors, and cytoplasmic and nuclear accumulation of beta-catenin were found in 68 and 66% of tumors, respectively. Significant correlations between cytoplasmic beta-catenin accumulation and the depth of invasion, venous invasion and focal dedifferentiation were observed. Cytoplasmic beta-catenin accumulation was also found to be a useful predictor of hematogenous metastasis (hazard ratio = 8.94, p = 0.054), though neither a reduced cell membrane level nor nuclear accumulation of beta-catenin correlated with metastasis.

Evaluation of a whole-genome amplification method based on adaptor-ligation PCR of randomly sheared genomic DNA

High‐throughput genetic studies often require large quantities of DNA for a variety of analyses. Developing and assessing a whole‐genome amplification method is thus important, especially with the current desire for large‐scale genotyping in previously collected samples for which limited DNA is available. The method we have developed, called PRSG, is based on an adaptor‐ligation–mediated PCR of randomly sheared genomic DNA. An unbiased representation was evaluated by performing PCR on 2,607 exons of 367 genes, which are randomly distributed throughout the genome, on PRSG products of hundreds of individuals. An infrequent loss (<1%) of the exon sequence on the PRSG products was found. Out of 307 microsatellites on various chromosomes, 258 (84%) were amplified in both the PRSG product and an original DNA, whereas 49 (16%) microsatellites were lost only in the PRSG product. Array CGH analysis of 287 loci for measuring the relative gene copy number demonstrated that a low bias was detected. Moreover, this method was validated on 100–1,000 laser‐captured cells from paraffin‐embedded tissues. These data show that PRSG can provide a sufficient amount of genomic sequence for a variety of genetic analyses as well as for long‐term storage for future work.

Correlation between Laminin-5 γ2 Chain Expression and Epidermal Growth Factor Receptor Expression and Its Clinicopathological Significance in Squamous Cell Carcinoma of the Tongue

Recent investigations have revealed that growth factors may influence the invasive activity of tumor cells. Expression of laminin-5 γ2 chain (LN-5 γ2) and epidermal growth factor receptor (EGFR) in squamous cell carcinomas of the tongue in 104 patients with stage II, III, and IVA, B (excluding the cases with distant metastasis) was examined immunohistochemically to determine the correlation between the two molecules and the associations with the clinicopathological features of each tumor. LN-5 γ2 expression was clearly demonstrated in the cytoplasm and EGFR in the cell membranes of cancer cells. A significant increase in positivity for LN-5 γ2 was observed in tumors showing poor differentiation (p < 0.001), infiltrative growth (p < 0.001), and deep invasion (p = 0.038). In a multivariate analysis, increased positivity for LN-5 γ2 was an independent predictor of an unfavorable outcome (p < 0.001). A significant increase in positivity for EGFR was observed in tumors showing infiltrative growth (p = 0.032) and poor prognosis (p = 0.008). The LN-5 γ2 expression was correlated significantly with EGFR expression (p < 0.001). Patients with tumor positivity for both molecules showed the worst prognosis (p < 0.001). LN-5 γ2 overexpression and EGFR overexpression is evident in tumors showing infiltrative growth, suggesting that EGFR may influence the invasive activity of tumor cells through overexpression of LN-5 γ2.

Integrin α6β4 as a suppressor and a predictive marker for peritoneal dissemination in human gastric cancer

Abstract Background & Aims: Because alterations of integrin expression in cancers contribute to cancer cell biology, we analyzed the association between the potential for peritoneal dissemination and integrin expression. Methods: The dissemination potential of 10 human gastric cancer cell lines in mice with severe combined immunodeficiency (SCID) was compared with the expression of various integrins. The relationship between integrin expression and peritoneal dissemination was also investigated in surgically resected gastric cancer cases. Results: The level of integrin β4 subunit expression was inversely correlated with dissemination potential. Introduction of a full-length complementary DNA (cDNA) for β4 subunit into cancer cells showing negligible β4 subunit expression markedly suppressed peritoneal dissemination and inhibition of endogenous integrin α6β4 by introduction of a cytoplasmic domain-deleted β4 subunit cDNA into cells showing high expression of β4 subunit promoted peritoneal dissemination. Apoptosis, which was histologically evident in peritoneal nodules of SCID mice, was induced in the cells with high β4 subunit expression by attachment to laminin and stimulation with growth factors in vitro. An immunohistochemical study of specimens from 120 cases of primary gastric cancer showed that patients with β4 subunit–positive tumors exhibited peritoneal dissemination only infrequently ( P P Conclusions: These results indicate that integrin α6β4 is both a suppressor and a predictive marker for peritoneal dissemination in gastric cancer. GASTROENTEROLOGY 2000;118:497-506

Prognostic significance of dysadherin expression in tongue cancer: Immunohistochemical analysis of 91 cases

The E-cadherin-mediated cell adhesion system is frequently inactivated by multiple mechanisms and is involved in tumor progression in many types of cancer. Recently we have reported a novel cell membrane glycoprotein, dysadherin, which has an anti—cell–cell adhesion function and downregulates E-cadherin. Expressions of dysadherin and E-cadherin were investigated immunohistochemically in 91 patients with squamous cell carcinoma of the tongue to determine the correlation between the 2 molecules and their associations with the clinicopathologic features of the tumors and with patient survival. Dysadherin was expressed at the cell membranes of many cancer cells. Twenty-five percent of the tumors showed dysadherin immunopositivity in more than 50% of the cancer cells. Sixty-nine percent of the tumors showed reduced E-cadherin immunopositivity. There was an inverse correlation between dysadherin expression and E-cadherin expression (P = 0.0001). Increased dysadherin expression was significantly correlated with an infiltrative type of growth pattern (P = 0.001), high tumor-node-metastasis (TNM) stage (P = 0.024), and poor patient survival (P = 0.003). After adjusting for growth pattern, TNM stage, and other clinicopathologic features, increased dysadherin expression and reduced E-cadherin expression were both significant predictors of poor survival (P = 0.0006). Increased dysadherin expression is a significant indicator of poor prognosis in patients with squamous cell carcinoma of the tongue.

Laminin 5 β3 and γ2 chains are frequently coexpressed in cancer cells

Laminin 5 plays an important role in cell migration during tumor invasion and tissue remodeling. However, previous studies have not clarified whether laminin 5 β3 chain is coexpressed with laminin 5 γ 2 chain in cancer cells. The present immunohistochemical study investigated the distribution of the laminin 5 β3 and γ 2 chains in 20 cases of squamous cell carcinoma of the tongue and 17 cases of colorectal carcinoma. Laminin 5 β3 and γ 2 chains were expressed in the cytoplasm of tumor cells. Tumor cells at the cancer–stromal interface and at the invasive front frequently showed immunopositivity for laminin 5 β3 and γ 2 chains in both squamous cell carcinoma of the tongue and colorectal carcinoma. Furthermore, strong expressions of these two proteins were observed in cancer cells invading in a scattered manner. Laminin 5 β3 expression correlated significantly with laminin 5 γ 2 expression in 20 cases of squamous cell carcinoma of the tongue (P = 0.0002) and 17 cases of colorectal carcinoma (P < 0.0001). These results suggest that in squamous cell carcinoma of the tongue and colorectal carcinoma, laminin 5 γ 2 chain and β3 chain are both important in the invasiveness of cancer cells.

Expression of Cadherin-Catenin Cell Adhesion Molecules, Phosphorylated Tyrosine Residues and Growth Factor Receptor-tyrosine Kinases in Gastric Cancers

Tyrosine phosphorylation of β‐catenin, an intracytoplasmic E‐cadherin‐binding protein, has been shown to disrupt the cadherin‐mediated cell adhesion system in vitro. In order to investigate the relationships of expression and tyrosine phosphorylation of cadherin‐catenin molecules and expression of growth factor receptor‐tyrosine kinase with loose cell‐to‐cell adhesion, immunohistochemical staining for E‐cadherin, α‐ and β‐catenin, phosphorylated tyrosine residues and tyrosine kinase receptors, including c‐erbB‐2, epidermal growth factor‐receptor (EGF‐R), c‐met and K‐sam, in 17 undifferentiated‐ and 10 differentiated‐type human gastric cancers was performed. Loss or reduced expressions of E‐cadherin and α‐ and β‐catenin (11, 11, 10 cancers, respectively) were observed in the former, but not the latter. Diffuse cytoplasmic staining of E‐cadherin, α‐ and β‐catenin and phosphotyrosine residues was observed frequently in the undifferentiated‐type cancers. The cytoplasmic localization of phosphotyrosine residues in undifferentiated‐type cancers was correlated significantly with K‐sam expression (P<0.01) and diffuse cytoplasmic staining of E‐cadherin (P<0.05) and β‐catenin (P<0.05). Expression of K‐sam protein was detected significantly more frequently in undifferentiated‐ (6/17; P<0.05) than differentiated‐type adenocarcinomas whereas the converse applied to c‐erbB‐2 expression (8/10 of the latter, P<0.05). Tyrosine phosphorylation of β‐catenin was directly confirmed in the protein extracts of one undifferentiated‐type gastric cancer. These data indicate that alteration of tyrosine phosphorylation status associated with K‐sam expression may cause the cytoplasmic distribution of cadherin‐catenin molecules and loose cell‐cell adhesion in undifferentiated‐type gastric cancers.