Possible involvement of RUNX3 silencing in the peritoneal metastases of gastric cancers.

PURPOSE: Our previous results suggested that a lack of RUNX3 function contributed to human gastric carcinogenesis, but the role of RUNX3 in progression and metastasis remains unclear. We examined RUNX3 expression in clinical samples of peritoneal metastases in gastric cancers. Changes in metastatic potential were assessed in animal experiments using stable RUNX3 transfectants of gastric cancer cells. Finally, global expression changes were analyzed using a cDNA microarray. EXPERIMENTAL DESIGN AND RESULTS: Significant down-regulation of RUNX3 through methylation on the promoter region was observed in primary tumors (75%) as well as in all clinical peritoneal metastases of gastric cancers (100%) compared with normal gastric mucosa. Stable transfection of RUNX3 inhibited cell proliferation slightly, and modest transforming growth factor-beta (TGF-beta)-induced antiproliferative and apoptotic effects were observed. Interestingly, it strongly inhibited peritoneal metastases of gastric cancers in animal model (P < 0.01). Furthermore, we did globally analyzed expression profiles of approximately 21,000 genes in parent cells and stable transfectant of RUNX3 using a cDNA microarray. Microarray analysis identified approximately 28 candidate genes under the possible downstream control of RUNX3, some of these genes were considered to be possibly involved in peritoneal metastases, which were related to signal transduction (vav3, TOLL-like receptor, MAPKK, MET, S1 00A1 1, and cathepsin E), apoptosis (caspase 9), immune responses (CD55 and TLR1O), and cell adhesion (sialyltransferase 1 and galectin 4). Some of the genes are involved in the TGF-beta signaling pathway. CONCLUSION: These results indicate that silencing of RUNX3 affects expression of important genes involved in aspects of metastasis including cell adhesion, proliferation, apoptosis, and promoting the peritoneal metastasis of gastric cancer. Identification of such genes could suggest new therapeutic modalities and therapeutic targets.     

E-cadherin expression in primary carcinomas of the breast and its distant metastases

Introduction  

Aberrant expression of E-cadherin has been associated with the development of metastases in patients with breast cancer. Even though the expression of E-cadherin has been studied in primary breast tumors, little is known about its expression at the distant metastatic sites. We investigate the relationship between E-cadherin expression in primary breast carcinoma and their distant, non-nodal metastases.     

Increased level of circulating adhesion molecules in the sera of breast cancer patients with distant metastases

The adhesion of circulating cancer cells to the vascular endothelium is an important at step in the hematogenous metastasis of cancer. E-selectin expressed on endothelial cells and carbohydrate ligands expressed on cancer cells mediate this adhesion. We investigated the clinical significance of such cell adhesion molecules in breast cancer. The cytosol concentration of sialyl Lewis(x) was found more elevated in cancerous tissue than that in adjacent non-cancerous tissue. In the serum, sialyl Lewis(x) and soluble E-selectin were seen elevated in patients with advanced and recurrent breast cancer, especially in those with distant metastases. From the above, we have concluded that sialyl Lewis(x) and soluble E-selectin could be used as tumor markers with a close relationship to the metastasis of breast cancer.      

Expression of the E-cadherin-catenin cell adhesion complex in primary squamous cell carcinomas of the head and neck and their nodal metastases.

Reductions in cell-cell adhesion and stromal and vascular invasion are essential steps in the progression from localized malignancy to metastatic disease. In this study, changes in the expression of the components of the E-cadherin-catenin cell adhesion complex have been investigated using immunohistochemical techniques in primary tumours and nodal metastases from 36 patients with squamous cell carcinoma of the head and neck. For 14 patients the corresponding primary and nodal metastases samples were available. None of the 51 samples showed normal E-cadherin expression when compared with either the adjacent normal squamous epithelium or with normal colonic epithelium that was used as positive control material. In 88% of primary tumours fewer than 50% of cells exhibited normal membranous E-cadherin expression. Loss of membranous E-cadherin expression was more extensive in poorly differentiated carcinomas while, in individual carcinomas, membranous E-cadherin expression was stronger in those parts of the neoplasm that expressed the differentiation marker involucrin. Expression of beta-catenin generally paralleled that of E-cadherin, but in 12 cases there was strong membranous beta-catenin expression in samples that exhibited predominantly cytoplasmic E-cadherin labelling. Expression of alpha-catenin was generally weak and did not correlate with the expression of either beta-catenin or E-cadherin. Marked intratumoral heterogeneity for protein expression was evident for all antibodies, and the abnormal expression of the catenins is a novel finding. E-cadherin is expressed more intensely in cells with greater squamous differentiation, but there was no correlation between the decreased expression of any of the adhesion molecules of the E-cadherin complex tested and local recurrence, metastasis or survival. The loss of expression of components of the E-cadherin complex is a common abnormality in squamous carcinomas and, while it may be permissive for metastasis, it does not appear to be the only determinant of this process.     

食管鳞癌细胞粘附分子的表达及其临床意义

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Expression of leukocyte adhesion molecules (ICAM-1/LFA-1) related to clinical behaviour in B cell lymphomas.

We investigated the expression of adhesion molecules of lymphocyte function-associated antigen-1 alpha (LFA-1 alpha) and its ligand intercellular adhesion molecule-1 (ICAM-1) on 74 well-characterized B cell lymphomas. The LFA-1 was expressed on B cell lymphomas (21/74; 28 per cent), but to a lesser degree than ICAM-1 which was highly expressed (48/74 cases; 64 per cent). From the results of bone marrow examination of 39 cases with B cell lymphomas, 13 of 16 cases with a lack of ICAM-1 molecule showed a higher incidence of marrow involvement, but nine of 23 cases with the expression of ICAM-1 molecule showed a lower incidence. These findings suggest that the lack of expression of the ICAM-1 molecule by B cell lymphomas correlates with bone marrow involvement by lymphoma cells (p < 0.05). Expression of the LFA-1 molecule appears not to correlate with marrow involvement (p < 0.05).     

The features of expression of cellular adhesion molecules in primary colorectal cancer and its metastases

Features of expression, distribution and interaction of E-cadherin, beta-catenin and CD-44v6 were immunologically assayed in samples from primary colorectal tumors (129) and their metastases to the lymph nodes (35) and liver (92).Reduction or total absence of E-cadherin expression was significantly more typical of tumors metastasizing, at different stages, to the liver (64/84, 76%) than in metastasis-free ones (14/45, 31%) (p=0.014). Enhanced cytoplasmic immunoreactivity and beta-catenin nuclear translocation occurred in 80% of disseminated tumors. Such alterations of E-cadherin, beta-catenin expression may be regarded as unfavorable prognosis factors involved in colorectal cancer. No significant correlation between CD-44v6 expression and tumor cell ability to metastasize was recorded.     

Expression of cell adhesion molecules and tumour infiltrating leucocytes in conjunctival melanoma

AIM: Very little is known about the immunology of conjunctival melanoma. We investigated the expression of cell adhesion molecules and the grade of tumour infiltration with lymphocytes and macrophages as important members for the communication between tumour cells and the immune system. METHODS: Archival material from 35 conjunctival melanomas was used for immunohistochemical detection of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), neural cell adhesion molecule (NCAM), CD3 and CD68 using monoclonal antibodies. Histological and clinical data for these tumours were assessed. RESULTS: ICAM-1 was expressed in 34 of 35 tumours; in 20 cases, more than 50% of the cells stained ICAM-1 positive. VCAM-1 was expressed in 21 of 34 tumours; in 17 cases, only a small proportion (1-25%) stained VCAM-1 positive. NCAM was expressed in 14 of 34 tumours; in 11 cases, only a small proportion (1-25%) stained NCAM positive. CD3-positive leucocytes were found in 26 of 32 tumours, whereas CD68-positive leucocytes were present in 33 of 34 tumours. Cox regression analysis revealed that patients with NCAM-positive tumours had a 6.4-fold higher risk of dying from conjunctival melanoma (P = 0.02). NCAM-positive tumours were preferentially (P = 0.03) located in prognostically 'unfavourable' areas (i.e. fornices, palpebral, caruncle) and had no or only a weak CD3-positive infiltrate (P = 0.03). CONCLUSIONS: ICAM-1, VCAM-1 and NCAM are differentially expressed in conjunctival melanoma. Leucocytes were present in almost every tumour. The association between NCAM expression and prognosis may be related to the differential anatomical tumour location of NCAM-positive and NCAM-negative tumours, and should be considered a preliminary observation due to the limited statistical power of this study.     

Increased expression of stromelysin-3 in basal cell carcinomas

We examined the expression of two groups of matrix metalloproteinases (MMPs), stromelysin and interstitial collagenase, in human skin cancer by northern blot analysis and in situ hybridization. Stromelyins-3 (ST-3) mRNA was overexpressed more than tenfold in 17 of 19 (89%) specimens of basal cell carcinoma (BCC) but in only three of 13 (23%) cutaneous squamous cell carcinomas (SCCs). Stromelysin-1 and -2 (ST-1/2) mRNA was overexpressed in three of 19 (16%) BCC and three of 13 (23%) SCC. Collagenase mRNA was overexpressed in nine of 19 (47%) BCC and three of 13 (23%) SCC. No mRNA for ST-3, ST-1/2, or collagenase was detected by northern analysis in 21 specimens of adjacent normal skin. Because of these findings, we examined the specific location of the ST-3 mRNA in BCC specimens by in situ hybridization. ST-3 mRNA was particular abundant in the characteristic stroma adjacent to the invasive basaloid tumor islands of the BCC and absent in the malignant cells. Moreover, ST-3 mRNA was expressed and induced by phorbol ester treatment in adult dermal fibroblasts but not in keratinocytes. In vitro studies have shown that MMPs are involved in the degradation of extracellular matrix molecules. Our finding of ST-3 mRNA overexpression in 17 of 19 (89%) BCC specimens is consistent with a role for this molecule in local invasion of stroma by BCC. Our in situ hybridization data suggested that while ST-3 is not expressed by malignant basal cells themselves, these tumor cells may induce the expression of ST-3 in adjacent nonmalignant stromal elements such as fibroblasts. © 1994 Wiley-Liss, Inc.     

Alterations in the expression of a hepatocyte cell adhesion molecule by transplantable rat hepatocellular carcinomas

Alterations in the expression of normal cell surface components on 13 transplantable hepatocellular carcinomas were examined using a heteroantiserum [anti-Mr 105,000 glycoprotein (gp 105)] reactive with a family of nine wheat germ agglutinin binding components from normal rat hepatocytes with an average molecular weight of 105,000. Analysis by two-dimensional polyacrylamide gel electrophoresis of components immunoprecipitated by anti-gp105 antiserum from detergent extracts of transplantable hepatocellular carcinoma cells surface labeled with 125I revealed qualitative and quantitative changes in the expression of anti-gp105-reactive components with the most consistent change being the apparent loss of a pair of acidic (pl 4.1 to 4.3) glycoproteins by all 13 transplantable hepatocellular carcinoma lines. One-dimensional peptide maps of fragments produced following digestion with V8 protease indicated that these acidic components were closely related in structure but differed significantly from other anti-gp105-reactive components. Immunodepletion analysis with monoclonal antibodies and heteroantisera reactive with individual components recognized by anti-gp105 antiserum showed that the two acidic glycoproteins were antigenically and structurally identical to cell-CAM 105, a Mr 105,000 glycoprotein involved in cell-cell adhesion of rat hepatocytes. Antibodies raised against purified cell-CAM 105 were specific in immunoprecipitation assays for the acidic components, strongly inhibited reaggregation of hepatocytes, and displayed no reactivity by indirect immunofluorescence or immunoprecipitation analysis with transplantable hepatocellular carcinoma cells. These results suggest that major alterations in the expression of cell-CAM 105 may be a consistent feature of the malignant phenotype.     

3种黏附分子表达与老年胃癌临床病理特征和预后的关系

目的:探讨黏附分子唾液酸化路易斯-X(sialyl Lewis-X,SLeX)、CD44v6和E-钙黏附素(E-cad-herin,E-Cad)蛋白表达与老年胃癌临床病理特征和患者预后的关系。方法:应用免疫组化技术检测48例老年胃癌中SLeX,CD44v6和E-Cad蛋白表达,并结合肿瘤的病理学行为和临床随访资料进行分析。结果:在老年胃癌组织中,SLeX,CD44v6和E-Cad表达阳性率分别为79.2%(38/48)、72.9%(35/48)和47.9%(23/48)。SLeX和CD44v6高表达和E-Cad低表达均与老年胃癌浆膜浸润、淋巴结转移和患者预后呈正相关(P<0.05)。结论:SLeX,CD44v6和E-Cad表达与老年胃癌转移和患者生存期密切相关,SLeX,CD44v6和E-Cad蛋白表达可作为判断老年胃癌预后的参考指标。     

Prognostic significance of combined expression of MUC1 and adhesion molecules in advanced gastric cancer

The objective of the present study was to evaluate the combination of MUC1 and the status of adhesion molecules in advanced gastric cancers as a possible predictor of patient survival. Two hundred and two paraffin-embedded specimens of gastric carcinoma were examined by immunohistochemical staining using monoclonal antibodies against MUC1 mucin, E-cadherin and beta-catenin. The expression of MUC1 was considered positive if at least 10% of the neoplastic cells were stained. E-cadherin and beta-catenin were classified into four groups. Only a membranous pattern, which was stained as strongly as normal epithelial cells, was judged as normal. The absent pattern (loss of staining), cytoplasmic pattern (cytoplasmic staining with loss of membranous expression), and heterogeneous pattern (cytoplasmic staining with preservation of membranous expression) were considered abnormal. There was a significant relationship between MUC1-positive expression and abnormal expression of E-cadherin (P=0.017). The cancer with abnormal E-cadherin expression or MUC1-positive expression increased, indicating that the cancer invasion was deep. Survival analysis of the outcome revealed that the survival time for those with abnormal E-cadherin/MUC1-positive expression was shorter than for those with other expression patterns. Multivariate analysis revealed that patients with abnormal E-cadherin/MUC1-positive expression had a poorer prognosis with significance (P<0.0001). In conclusion, abnormal E-cadherin/MUC1-positive expression pattern in advanced gastric cancer is an independent unfavorable prognostic marker.     

Galectin 3 expression in primary oral squamous cell carcinomas

Background

Immunologic factors can promote the progression of oral squamous cell carcinomas (oscc). The phylogenetic highly conserved protein Galectin 3 (Gal3) contributes to cell differentiation and immune homeostasis. There is evidence that Gal3 is involved in the progression of oscc and influences the regulation of macrophage polarization. Macrophage polarization (M1 vs. M2) in solid malignancies like oscc contributes to tumor immune-escape. However, the relationship between macrophage polarization and Gal3 expression in oscc is not yet understood. The current study analyzes the association between histomorphologic parameters (T-, N-, L- Pn-status, grading) and Gal3 expression resp. the ratio between Gal3 expressing cells and CD68 positive macrophages in oscc specimens.

Methods

Preoperative diagnostic biopsies (n?=?26) and tumor resection specimens (n?=?34) of T1/T2 oscc patients were immunohistochemically analyzed for Gal3 and CD68 expression. The number of Gal3 expressing cells and the ratio between CD68 and Gal3 expressing cells was quantitatively assessed.

Results

In biopsy and tumor resection specimens, the number of Gal3 positive cells as well as the Gal3/CD68 ratio were significantly (p?<?0.05) higher in T2 oscc compared to T1 cases. In biopsy specimens, a significantly (p?<?0.05) increased Gal3 expression and Gal3/CD68 ratio was associated with the progression marker lymph vessel infiltration (L1). Tumor resection specimens of cases with lymph node metastases (N+) had a significantly (p?<?0.05) increased Gal3 expression. Additionally, a high Gal3/CD68 ratio correlated significantly (p?<?0.05) with higher grading (G3) in tumor resection specimens.

Conclusion

High Gal3 expression in oscc is associated with tumor size (T-status) and parameters of malignancy (N-, L-status, grading). Gal3 might contribute to M2 macrophage mediated local immune tolerance. Gal3 expression shows association with prognosis in oscc and represent a potential therapeutic target.

     

Expression of cell adhesion molecules and chemokine receptors: angioinvasiveness in nasal NK/T-cell lymphoma

Sinonasal natural killer (NK)/T-cell lymphoma (NKTCL) is closely associated with Epstein-Barr virus (EBV) infection and expresses latent membrane protein (LMP)-1 and EB nuclear antigen (EBNA)-1, i.e., latency II of EBV infection. Angioinvasion by neoplastic cells is a characteristic feature of NKTCL, but its mechanism is unknown. To elucidate the molecular mechanism of angio-invasiveness in NKTCL, expression of cell adhesion molecules and chemokine receptors at mRNA and protein levels was examined using real-time PCR and immunohistochemistry in 17 NKTCL together with 10 diffuse large B-cell lymphoma (DLBL) and 9 non-neoplastic nasal mucosa as controls. EBV DNA was detected in 14 of 16 NKTCL examined, and 7 of these 14 expressed LMP-1. mRNA expression levels of integrin subunits alpha4, alpha L, alpha M, and beta2 were significantly higher in NKTCL than non-neoplastic controls. Integrin subunits alpha2 and alpha M were expressed at a significantly higher level in NKTCL with angioinvasion than those without. Expression level of alpha M was significantly higher in 7 cases of NKTCL with LMP-1 expression than 7 without. Immunohistochemistry showed expression of these molecules in NKTCL cells. These findings suggest that EBV infection might be involved in the pathogenesis of angioinvasion of NKTCL through up-regulation of alpha M by LMP-1.     

EGFR and HER3 mRNA expression levels predict distant metastases in locally advanced rectal cancer

Aberrant activation of the HER signaling pathways plays a critical role in the invasive and metastatic potential of tumors. The aim of this study was to address whether, in rectal cancer, alterations of these pathways could have a value as prognostic factors to be used to identify patients who are at risk of distant metastases. Therefore, the mRNA expression of the four members of the HER family as well as the frequency of PTEN allelic loss and KRAS/BRAF mutations were determined in pretreatment biopsies from a series of 100 locally advanced rectal cancers and then their ability to predict distant metastases was evaluated. Over‐expression of EGFR (p = 0.021), HER2 (p = 0.011) and HER3 (p = 0.020) was significantly associated with worse metastasis‐free survival in univariate analysis. In multivariate analysis, both over‐expression of EGFR (p = 0.028) and HER3 (p = 0.011) remained independent prognostic factors for distant metastasis. In conclusion, quantification of EGFR and HER3 mRNA expression in pretreatment biopsies may be useful to identify patients who are at risk of developing metastases.     

Altered expression and mutation of beta-catenin gene in gastric carcinomas and cell lines

beta-catenin serves not only as a structural component of the E-cadherin-mediated cell-cell adhesion system, but also as a signaling molecule of the Wnt/wingless pathway. Deregulated expression of beta-catenin and mutations of the gene have been identified in a number of human malignancies. To determine the role of beta-catenin defects in stomach cancer, we investigated beta-catenin exon 3 mutations and altered protein expression in 77 primary gastric carcinomas and 11 cell lines. In addition, the immunohistochemical expression pattern of beta-catenin in 303 consecutive gastric cancers was determined and their relationships with clinicopathologic features and patient outcome were investigated. This study revealed 5% (4 of 77) tumors and 27% (3 of 11) cell lines with beta-catenin gene alteration, 6 missense mutations, and 1 interstitial deletion. These genetic changes were shown to correlate closely with nuclear localization of the protein (p = 0.001). In an immunohistochemical analysis, abnormal expressions of beta-catenin, such as nuclear accumulation and loss of membranous distribution, were detected in 27% (81 of 303) of tumors overall. These altered beta-catenin expressions were more commonly observed in 37% (58 of 158) diffuse type gastric carcinomas (p < 0.001). Loss of membranous beta-catenin staining was associated with poor survival (p = 0.045). In conclusion, our results demonstrate that beta-catenin mutations are common in gastric cancer cell lines but occur infrequently in gastric carcinoma tissues. These mutations are one of the causes of the nuclear accumulation of beta-catenin. Frequent abnormalities of beta-catenin expression in gastric carcinoma support the idea that both structural and signaling functions of the protein play a critical role in gastric carcinogenesis.     

Absence, or low expression, of leukocyte adhesion molecules CD11 and CD18 on Burkitt lymphoma cells

Leukocyte adhesion to cells is mediated by the cell-surface glycoprotein complex CD11a-c/CD18 and, in some cases, the glycoprotein gp84. The process is associated with leukocyte activation and modulates lymphocyte proliferation and maturation. Epstein-Barr virus (EBV)-transformed normal B lymphocytes give rise to lymphoblastoid cell lines (LCLs) which grow as large clusters mediated by adhesion molecules. In contrast, newly explanted EBV-infected Burkitt lymphoma (BL) cells usually grow as single cells or as loose clusters. We now report that EBV positive- and EBV-negative BL lines lack the adhesive protein complex, or have only low levels of it, whereas LCLs, representing various stages of B-lymphocyte development, contain considerably higher amounts, as measured by immunofluorescence flow cytometry and immunoprecipitation. The level of gp84 expression is of the same magnitude in both types of cells.     

Loss of caspase-1 gene expression in human gastric carcinomas and cell lines

The caspases are a family of aspartic acid-specific proteases that fulfill varied and often critical roles in mammalian apoptosis or in the proteolytic activation of cytokines. Caspase-1 (interleukin-1beta-converting enzyme) is a member of the cysteine protease family, which cleaves target proteins following aspartic acid residues. We investigated caspase-1 expression in stomach cancer, tissues and cell lines. Of 301 consecutive gastric carcinomas, 58 cases (19.3%) showed the expressional loss of caspase-1. Loss of caspase-1 expression was significantly associated with pTNM stage (p=0.03), lymph node metastasis (p=0.01) and patient survival (p<0.01). Caspase-1 expression was also significantly correlated in an inverse manner with p53 expression (p<0.01). Among the 11 gastric cancer cell lines examined, three cell lines showed loss of expression at the protein and mRNA levels. On treatment with 5-aza-2'-deoxycytidine (5-aza-C), and/or trichostatin A (TSA), all three cell lines re-expressed caspase-1 mRNA. The above findings suggest that epigenetic events such as DNA methylation and histone deacetylation play important roles in the regulation of caspase-1, and that loss of caspase-1 expression is associated with poor survival in gastric carcinoma.