Tetsu Yamane

EGFR protein overexpression and gene amplification in squamous cell carcinomas of the esophagus

Overexpression of epidermal growth factor receptor (EGFR) is observed in many cancers, sometimes accompanied by gene amplification. Recently, several clinical therapies targeting EGFR were developed, but the eligibility criteria for these therapies is not fully established. To develop such eligibility criteria for esophageal squamous cell carcinoma (ESCC), we sought to clarify: (i) the exact frequency of EGFR overexpression, (ii) the relationship between protein overexpression and gene amplification, (iii) the relationship between gene amplification and specific gene mutations and (iv) the correlation between the status of EGFR and clinical or pathological features. Immunohistochemistry revealed that EGFR protein is overexpressed in 53 (50%) of the 106 ESCC examined. Fluorescence in situ hybridization (FISH) indicated clear EGFR gene amplification in 15 of the 53 tumors, somewhat higher EGFR copy in 32 cases, and no increase in 6 cases. Gene amplification was significantly associated with high level overexpression. Direct sequencing of exons 19 and 21 of EGFR revealed no mutations in 15 tumors exhibiting gene amplification, and no mutations in 25 tumors not exhibiting gene amplification. Overexpression of EGFR was significantly correlated with depth of invasion of the tumor.In conclusion, anti‐EGFR therapies may be appropriate for patients with ESCC. We assume that combined analyses by immunohistochemistry/FISH would clarify aberrations in protein and gene function, and could help to identify those patients who may benefit from anti‐EGFR therapy.

Epigenetic silencing of TTF-1/NKX2-1 through DNA hypermethylation and histone H3 modulation in thyroid carcinomas.

Thyroid transcription factor-1 (TTF-1), also known as NKX2-1, is a homeodomain containing transcriptional factor identified in thyroid, lung and central nervous system. In the thyroid, TTF-1 is essential for thyroid organogenesis and governs thyroid functions by regulating various thyroid-specific genes. We previously demonstrated that most differentiated thyroid neoplasms, including follicular adenomas/carcinomas and papillary carcinomas, express TTF-1 at both protein and mRNA levels. However, certain subtypes of thyroid cancers have shown low or negative expression of TTF-1. The aim of our study was to investigate the function of epigenetic modification in dysregulation of TTF-1 in thyroid carcinoma cells. We evaluated the expression of TTF-1 in primary thyroid tissues (normal thyroid, papillary carcinoma and undifferentiated carcinoma) and in thyroid carcinoma cell lines using immunohistochemistry and RT–PCR. Methylation-specific PCR targeting CpG islands of TTF-1 and chromatin immunoprecipitation (ChIP) for histone H3 lysine 9 (H3-lys9) were applied to clarify the correlation of the TTF-1 expression profile and epigenetic status. We also explored whether epigenetic modifiers, including 5-aza-deoxycytidine, could restore TTF-1 expression in thyroid carcinoma cells. In our current study, immunohistochemistry and RT–PCR showed positive expression of TTF-1 in normal thyroids and papillary carcinomas. Meanwhile, most of the undifferentiated carcinomas and the cell lines lost TTF-1 expression. No methylation in the CpG of TTF-1 promoter was detected in normal thyroids or papillary carcinomas. In contrast, DNA methylation was identified in 60% of the undifferentiated carcinomas (6/10) and 50% of the cell lines (4/8). ChIP assay demonstrated that acetylation of H3-lys9 was positively correlated with TTF-1 expression in thyroid carcinoma cells. Finally, DNA demethylating agents could restore TTF-1 gene expression in the thyroid carcinoma cell lines. Our data suggest that epigenetics is involved with inactivation of TTF-1 in thyroid carcinomas, and provide a possible means of using TTF-1 as a target for differentiation-inducing therapy through epigenetic modification.

The Expression of a Type II Transmembrane Serine Protease (Seprase) in Human Gastric Carcinoma

Objective: The invasion and metastasis of carcinoma cells require the proteolytic degradation of the extracellular matrix by various cell surface proteases. Among these, seprase is a type II transmembrane serine protease absent in normal tissues and it has been implicated in the invasion of the extracellular matrix by both tumor and stromal cells in human breast carcinoma and melanoma. In the present study, the expression of seprase mRNA, protein and its gelatin-degrading activity in human gastric carcinoma were examined to substantiate the potential role of seprase in gastric carcinoma invasion. Methods: We have examined the seprase expression in human gastric carcinoma (n = 34) by RT-PCR, Western immunoblotting analysis, immunohistochemistry, and gelatin zymography. Results: Immunoblotting analysis using mAb D8 directed against seprase showed that the carcinoma tissues in 26 out of 34 cases of gastric cancer expressed a dimeric form of seprase but their normal counterparts did not. Gelatin zymography confirmed that the isolated seprase exhibited the gelatin-degrading activity and was active. Seprase-expressing carcinoma tissues were more often found in the scirrhous type than in other types of gastric carcinoma. RT-PCR analysis showed that seprase mRNA was present in carcinoma tissues but not in normal tissues. Immunohistochemically, seprase was mainly located in gastric carcinoma cells, weakly in stromal cells and microvessel endothelial cells in the tumor nest, and none in normal cells. Conclusions: Our studies showed the unique expression and localization of seprase in the tumor and stromal cells within human gastric carcinoma but not in normal tissues, suggesting a role of seprase in the invasive and metastatic progression of gastric carcinoma.

Protein overexpression and gene amplification of c-erb B-2 in pulmonary carcinomas: a comparative immunohistochemical and fluorescence in situ hybridization study.

Amplification of the c-er b B-2 gene (located on 17q11.2–12) is accompanied by overexpression of its cell surface receptor product, p185ERBB2. In pulmonary carcinomas, however, there has been disagreement between the reported frequencies of gene amplification and overexpression. To clarify their relationship, the correlation between the cellular expression of p185ERBB2 and the level of c-erbB-2 gene amplification was studied. A total of 195 pulmonary carcinomas (182 primary and 13 metastatic) were examined immunohistochemically using a polyclonal antibody, which recognizes the internal domain of the human c-erbB-2 protein, and positive tumors were further examined for the gene amplification by dual-color fluorescence in situ hybridization using probes for centromere 17 and 17q11.2–12. By immunohistochemistry, distinct membrane staining was found in an adenocarcinoma, a large cell carcinoma and a metastatic carcinoma from the breast, and cytoplasmic and/or faint membranous staining was observed in 23 non-small cell lung carcinomas. It was in the two primaries and the metastatic carcinoma that more than 8-fold amplification of c-erbB-2 was found by fluorescence in situ hybridization. Especially, in the two primary carcinomas, tumor cells had amplified genes with the signals forming one or two clusters, indicating that the amplified gene was present in homogeneously staining regions. Among the 23 tumors, three tumors showed low-level amplification (less than 3-fold), which was differentiated from polysomy 17 found in the other two. In the 30 non-small cell lung carcinomas selected at random from 151 with negative immunostaining, there were five trisomy 17, but no tumors with the gene amplification. This suggests that although c-erbB-2 amplification in pulmonary carcinoma is rare, it occurs in the form of a homogeneously staining region and is thought to control the overexpression of the protein in the cell membrane. New adjuvant therapy using a humanized antibody to the oncoprotein may be beneficial to patients with these tumors.

Hypoxia-inducible adenosine A2B receptor modulates proliferation of colon carcinoma cells

Extracellular adenosine regulates a wide variety of physiological processes by interacting with 4 adenosine receptor subtypes: A1, A2A, A2B, and A3. However, little is known of their pathophysiological roles in human cancers. In this study, we examined the expression pattern of adenosine receptors in various colorectal tissues and human colon carcinoma cell lines and investigated the biologic functions regarding colon carcinogenesis. Using reverse transcriptase polymerase chain reaction and Western blotting, we found that adenosine receptor A2B (ADORA2B) was consistently up-regulated in colorectal carcinoma tissues and colon cancer cell lines compared with normal colorectal mucosa. In immunohistochemistry, we observed diffuse immunopositivity of ADORA2B in 67% of colorectal adenocarcinomas (39/58), 17% of tubular adenomas (5/30), and 0% of normal colon glands (0/62). During a hypoxic state, there was also a significant induction of ADORA2B expression in the messenger RNA level at 8 hours of incubation and in the protein level at 24 hours of incubation in colon carcinoma cell lines. To examine the function of ADORA2B, we applied an ADORA2B-selective antagonist (MRS1754) to the colon carcinoma cells, which significantly inhibited cell growth in a dose-dependent manner as demonstrated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay. In conclusions, ADORA2B was overexpressed in colorectal carcinomas grown under a hypoxic state, presumably promoting cancer cell growth. Our data suggest that this adenosine receptor is a potential therapeutic target for colorectal cancer.

Global histone modification of histone H3 in colorectal cancer and its precursor lesions

Chromatin remodeling through histone modification is an important mechanism of epigenetic gene dysregulation in human cancers. However, little is known about global alteration of histone status during tumorigenesis and cancer progression. Histone H3 status was examined in benign and malignant colorectal tumors by immunohistochemistry and Western blotting. For immunohistochemical evaluation, 4 anti-histone H3 antibodies, specific to dimethylation at lysine 4 (H3K4me2), acetylation at lysine 9 (H3K9ac), dimethylation at lysine 9 (H3K9me2), and trimethylation at lysine 27 (H3K27me3), were used. On immunohistochemistry, H3K4me2, H3K9ac, and H3K27me3 showed no significant changes between normal and colorectal tumors. On the other hand, the global level of H3K9me2 was distinctly higher in neoplastic cells (adenoma and adenocarcinoma) than in normal glandular cells. In addition, it was significantly higher in adenocarcinoma than in adenoma. Correspondingly, Western blotting confirmed that H3K9me2 expression was significantly higher in adenocarcinomas than in normal colorectal mucosa. No alteration of H3K9me2 was observed with tumor differentiation and with the histological subtypes of colorectal cancers. These results suggest that aberration of the global H3K9me2 level is an important epigenetic event in colorectal tumorigenesis and carcinogenesis involved with gene regulation in neoplastic cells through chromatin remodeling.

Estimation of prognoses for cervical intraepithelial neoplasia 2 by p16INK4a immunoexpression and high-risk HPV in situ hybridization signal types

The present study used immunohistochemical staining and in situ hybridization (ISH) to examine whether progression of cervical intraepithelial neoplasia, grade 2 (CIN 2) can be predicted by p16INK4a immunoexpression and high-risk human papilloma virus (HPV) ISH signal types. We studied 52 cases histologically diagnosed with CIN 2: dysplasia regressed in 28 cases; 13 cases progressed to CIN 3; and CIN 2 persisted in 11 cases. Expression of p16INK4a and high-risk HPV signal both related to grade of CIN. Stronger p16INK4a immunoexpression and a higher frequency of expression of a punctate nuclear signal were observed in CIN 2 lesions before progression compared with those before regression. CIN 2 cases in which moderate to strong immunoexpression of p16INK4a and a punctate signal were observed simultaneously progressed to CIN 3 in 10 (91%) of 11 cases. CIN 2 cases with moderate to strong immunoexpression of p16INK4a and a high-risk HPV punctate signal should be treated because of the great risk of progression.

Laminar Shear Stress–Induced GRO mRNA and Protein Expression in Endothelial Cells

BACKGROUND The shear stress induced by blood flow may play a pivotal role in the induction or prevention of atherosclerosis by changing endothelial functions. To disclose the mechanisms of this change, we prepared an endothelial cell (EC) cDNA library to select specific clones expressed in response to shear stress. METHODS AND RESULTS The mRNA of cultured confluent bovine aortic ECs (BAECs) subjected to steady laminar shear stress (30 dyne/cm2) for 4 hours was separated, and a cDNA library was prepared. Nine clones whose expressions were specifically enhanced by the shear stress were selected by use of a differential hybridization method. One clone had 94% homology at the nucleotide sequence level to Oryctolagus cuniculus gro (GRO) mRNA and 79% homology at the amino acid sequence level to human GRO-beta. The GRO mRNA expression was increased in both BAECs and human umbilical vein ECs (HUVECs) after the ECs were subjected to high (30 dyne/cm2) and low (5 dyne/cm2) laminar shear stress. GRO-alpha and/or -beta protein expression also increased after the HUVECs and BAECs were subjected to shear stress. Because GRO protein has been shown to function as an adhesion factor of monocytes on the surface of ECs, we studied whether shear stress-induced monocyte adhesion was caused by GRO protein expression on ECs. The 4-hour shear stress enhanced monocyte adhesion to ECs by 2.5-fold over control levels, and this enhancement was inhibited by 53% by anti-GRO-alpha antibody. CONCLUSIONS The present study is the first report that shear stress induced the expression of GRO mRNA and protein in ECs and enhanced the monocyte adhesion on ECs via GRO protein. Further investigations of the functions and participation in atherogenesis of this selected clone may clarify the significance of shear stress on atherogenesis.

Seprase, a Membrane-Type Serine Protease, Has Different Expression Patterns in Intestinal- and Diffuse-Type Gastric Cancer

Objective: Seprase is an integral membrane serine proteinase with gelatinase activity that may be involved in cancer invasion and metastasis. However, the pathophysiologic significance of its expression in gastric cancer tissue has not been fully elucidated. Methods: Seprase expression and distribution in gastric cancer specimens obtained from 133 patients were examined by immunohistochemistry and immunoblotting. Results: Immunohistochemistry showed that in intestinal-type cancer, which includes well and moderately differentiated adenocarcinoma, seprase immunoreactivity was mainly recognized in the moderately differentiated cells and not in the well differentiated cells. In the diffuse type, which includes poorly differentiated adenocarcinoma and signet ring cell carcinoma, seprase immunoreactivity was seen mainly in cells with poor cell-to-cell junctions. The reactive pattern in the cells was different between moderately differentiated adenocarcinoma and diffuse-type carcinoma. Besides the cytoplasm, the cell membrane also apparently reacted in the former, while only the cytoplasm reacted diffusely in the latter. Seprase immunoreactivity was also recognized in endothelial cells and stromal cells especially adjacent to tumor nests. The immunoreactivity of the stromal cells was more abundant in the intestinal type than in the diffuse type, and these stromal expressions of seprase in the intestinal type correlated with the liver (13/13 = 100% of cases with metastases) or lymph node metastases (33/34 = 97% of cases with metastases). Immunoblotting showed that the levels of seprase protein were higher in intestinal-type cancer than in diffuse-type cancer. Conclusion: These results suggested that there is a difference in seprase expression between intestinal- and diffuse-type gastric cancer; this difference may reflect distinct biological features of these types of cancer.

Activation of EDTA-resistant gelatinases in malignant human tumors.

Among the many proteases associated with human cancer, seprase or fibroblast activation protein alpha, a type II transmembrane glycoprotein, has two types of EDTA-resistant protease activities: dipeptidyl peptidase and a 170-kDa gelatinase activity. To test if activation of gelatinases associated with seprase could be involved in malignant tumors, we used a mammalian expression system to generate a soluble recombinant seprase (r-seprase). In the presence of putative EDTA-sensitive activators, r-seprase was converted into 70- to 50-kDa shortened forms of seprase (s-seprase), which exhibited a 7-fold increase in gelatinase activity, whereas levels of dipeptidyl peptidase activity remained unchanged. In malignant human tumors, seprase is expressed predominantly in tumor cells as shown by in situ hybridization and immunohistochemistry. Proteins purified from experimental xenografts and malignant tumors using antibody- or lectin-affinity columns in the presence of 5 mmol/L EDTA were assayed for seprase activation in vivo. Seprase expression and activation occur most prevalently in ovarian carcinoma but were also detected in four other malignant tumor types, including adenocarcinoma of the colon and stomach, invasive ductal carcinoma of the breast, and malignant melanoma. Together, these data show that, in malignant tumors, seprase is proteolytically activated to confer its substrate specificity in collagen proteolysis and tumor invasion.