Yuji Hinoda

Contribution of matrilysin (MMP-7) to the metastatic pathway of human colorectal cancers

BACKGROUND/AIM Matrilysin is one of the matrix metalloproteinases that has a critical role in tumour invasion, and is often expressed in gastrointestinal cancers. The aim of this study was to examine the role of matrilysin in metastasis of human colorectal cancers. PATIENTS (SUBJECTS)/METHODS The relation between matrilysin expression and Dukes’s type was investigated immunohistochemically in 83 surgically resected colorectal cancers, including five with liver metastasis. Moreover, the effects of matrilysin on the in vivo invasive and metastatic potential of colon cancer cells transfected with matrilysin cDNA were examined after subcutaneous injection into SCID mice. RESULTS In 46% of primary and all of metastatic liver tumours, over 10% of cancer cells were stained positively for matrilysin. The expression of matrilysin correlated significantly with the presence of nodal or distant metastases (p<0.05). In addition, matrilysin transfectants formed invasive tumours and multiple liver metastases in SCID mice, without producing any significant difference in the subcutaneous tumour growth from mock transfectants. Casein zymography showed that the invading and metastasised tumours showed conspicuous matrilysin activity, which correlated with the number of metastatic lesions (p<0.001). CONCLUSIONS Matrilysin showed a correlation with metastasis in a cohort of 83 colorectal cancer patients and marked metastatic potentiation in human colorectal cancer xenografts, indicating that it may play a critical role in the metastatic pathway of colorectal cancers.

DISTINCT METHYLATION PATTERN AND MICROSATELLITE INSTABILITY IN SPORADIC GASTRIC CANCER

Aberrant 5′ CpG island methylation is an alternative mechanism of gene inactivation during the development of cancer as demonstrated for several tumor‐suppressor genes. Also, marked relationship of microsatellite instability (MSI) and DNA methylation has been reported in sporadic colorectal cancer, which is a result of epigenetic inactivation of hMLH1 in association of promoter hypermethylation. In the present study, we investigated the 5′ CpG island hypermethylation of hMLH1, E‐cadherin and p16 in 61 primary gastric cancers (GCs) by using combined bisulfite restriction analysis (COBRA) and methylation‐specific PCR (MSP), and their MSI status. Of 61 GCs investigated, 5 (8.1%) tumors presented hMLH1 methylation, 16 (26.2%) and 25 (40.9%) showed E‐cadherin and p16 methylation respectively, and 8 (13.1%) presented high‐frequency MSI (MSI‐H). Of the 8 MSI‐H patients, 5 presented hMLH1 methylation, whereas no low‐frequency MSI (MSI‐L) and microsatellite stable (MSS) cases exhibited hMLH1 methylation (5/8 vs. 0/43, p < 0.00001). Furthermore, these patients also presented E‐cadherin and p16 hypermethylation. Our data showed a significant correlation between hMLH1 methylation and MSI in GC, and suggested that a common mechanism of aberrant de novo methylation can be postulated in these cancers. Int. J. Cancer 83:309–313, 1999.

Relation of enhanced secretion of active matrix metalloproteinases with tumor spread in human hepatocellular carcinoma

BACKGROUND & AIMSnMatrix metalloproteinases have been implicated in invasion and metastasis of various human malignant tumors, but its role in human hepatocellular carcinoma has not been characterized in detail. The aim of the present study was to examine the secretion and activation of metalloproteinases in liver tissues from patients with hepatocellular carcinoma and evaluate its relationship with clinicopathologic characteristics.nnnMETHODSnActivity of metalloproteinases was measured in 30 surgical specimen pairs of human primary hepatocellular carcinoma and adjacent nontumoral liver and in five cultured human hepatoma cell lines using zymography. A comparison of this activity with clinicopathological features was made.nnnRESULTSnIn the liver tissues, enhanced secretion of active forms of gelatinase A and matrilysin was associated with portal venous invasion (P < 0.05, respectively), intrahepatic metastasis (P < 0.05, respectively), and recurrence within the first postoperative year (P < 0.01 and P < 0.05, respectively). Enhanced messenger RNA expression for membrane type 1-matrix metalloproteinase was observed in 22 of 30 cases and associated with capsule invasion and the activation of progelatinase A (P < 0.05, respectively).nnnCONCLUSIONSnActive gelatinase A, active matrilysin, and membrane type 1-matrix metalloproteinase may play an important role in tumor spread of human hepatocellular carcinoma.

EXPRESSION OF CD10/NEUTRAL ENDOPEPTIDASE IN NORMAL AND MALIGNANT TISSUES OF THE HUMAN STOMACH AND COLON

The expression of common acute lymphoblastic leukemia antigen (CALLA; CD10), which is identical to neutral endopeptidase (NEP, EC3.424.11), was examined in the malignant and adjacent noninvaded tissues of the human stomach and colon (n = 27). All of 27 normal and 18 well or moderately differentiated adenocarcioma tissue specimens were positive for monoclonal antibody (mAb) NL-1 against CD10/NEP, whereas the expression level was clearly decreased in all of 9 specimens of poorly differentiated adenocarcinoma. In addition, all of 7 gastric or colorectal carcinoma cell lines tested showed decreased expression of CD10/NEP. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the crude antigen J5 from the normal colon tissue lysate by mAb J5 detected a single band of approximately 100 kDa that was consistent with that of NALM-6 cells used as a positive control. These findings suggest that CD10/NEP is expressed in normal epithelial cells of the human stomach and colon, whereas the expression level is decreased in the poorly differentiated type of adenocarcinoma.

Reduced Invasive and Metastatic Potentials of KAI1‐transfected Melanoma Cells

KAI1 is a metastasis suppressor gene for human prostate cancer. To reveal the effect of KAI1 on the in vivo metastasis of tumors other than prostatic cancer, we transfected a human KAI1 cDNA into highly metastatic B16‐BL6 murine melanoma cells and established stable transfectant clones with different expression levels of KAI1 message. The following results were obtained with the use of those transfectants. (1) Cell aggregation assay revealed a significantly enhanced Ca2+‐independent aggregation of B16‐BL6 cells by KAI1 cDNA transfection compared with mock transfectants (P<0.01). (2) The in vivo phagokinetic activity and invasive ability of KAI1 transfectants were clearly decreased as compared with those of mock transfectants (P<0.01). There was no significant effect of KAI1 expression on the in vitro or in vivo proliferation of B16‐BL6 cells. (3) Lung colony formation of intravenously injected KAI1 transfectants in nude mice was significantly reduced as compared with mock transfectants or parental B16‐BL6 cells (P<0.01). These data suggest that KAI1 expression gives rise to the suppression of invasive and metastatic potentials of B16‐BL6 cells.

Overexpression of cyclooxygenase‐2 protein is less frequent in gastric cancers with microsatellite instability

Overexpression of cyclooxygenase‐2 (COX‐2) has been reported in gastric cancers. However, the relationship between expression of COX‐2 and clinico‐pathological or genotypic features has not been elucidated. To address the issue, expression of COX‐2 protein was analyzed in 100 gastric cancers as well as 7 gastric cancer cell lines by using immunoblot analysis. Overexpression of COX‐2 in cancer tissues compared with matched non‐cancerous tissues was found in 70% of cases and was significantly associated with lymphatic involvement, lymph node metastasis and advanced tumor stage. Interestingly, overexpression of COX‐2 was less frequent in gastric cancers with microsatellite instability (MSI) than in those without MSI (8/20 vs. 62/80, p < 0.01). Expression of COX‐2 protein was detected in some gastric cancer cell lines without MSI at various levels, but not in those with MSI. Our results suggest that overexpression of COX‐2 may play an important role in tumor progression of gastric cancer and also support the notion that gastric cancers with and without MSI represent distinctive pathways of carcinogenesis. We also observed a reduction of MSI phenotype after aspirin or sulindac treatment in a hMLH1‐defective gastric cancer cell line SNU‐1, which lacks COX‐2 expression. Int. J. Cancer (Pred. Oncol.) 84:400–403, 1999.

Expression of c-kit and kit Ligand in Human Colon Carcinoma Cells

To determine whether c-kit and kit ligand (KL) mRNAs could be expressed in human epithelial tumors, reverse transcriptase-polymerase chain reaction and Northern blot analysis were performed. KL mRNA was shown to be expressed in a variety of epithelial tissues and cell lines. The expression of c-kit mRNA was then examined in hepatocellular and colon carcinoma cell lines. While hepatocellular carcinoma cell lines did not express c-kit mRNA as far as we could ascertain, 2 of 5 colon carcinoma cell lines showed the expression of both c-kit and KL mRNAs. Furthermore, the expression of c-kit in these cells was demonstrated at the protein level by flow cytometry. These data suggest that c-kit and KL may play an important role as an autocrine loop in the proliferation of some colon carcinoma cells.

Detection of circulating anti-MUC1 mucin core protein antibodies in patients with colorectal cancer.

Abstract: MUC1 mucin has a unique immunogenic peptide epitope in the extracellular domain, which has been shown to induce humoral and cellular immune response. In this study, we evaluated the pathophysiological significance of circulating anti-MUC1 mucin core protein IgG antibodies (anti-MUC1 antibodies) in colorectal cancer by Western blot analysis and 51Cr release assay. Anti-MUC1 antibodies were detected in 5 of 31 (16.1%) healthy subjects and in 27 of 56 (48.2%) patients with colorectal cancer. The presence of circulating anti-MUC1 antibodies was not significantly correlated with the level of circulating antigen MUSE11 or with other clinicopathological parameters tested. The incidence of positivity for anti-MUC1 antibodies in stage I and II (staged according to the General Rules for Clinical and Pathological Studies on Cancer of the Colon and Rectum of the Japanese Research Society for Cancer of the Colon and Rectum) cancers was 45.5% and 58.8%, respectively, suggesting that positivity for these antibodies may be of use as an adjunct for the diagnosis of colorectal cancer in the early stages in the absence of serious complications such as liver diseases. Because of the epitope similarity, anti-MUC1 antibodies in the serum may function in a manner similar to that of anti-MUC1 peptide monoclonal antibodies (mAbs). We therefore observed antibody-dependent cell mediated cytotoxicity with anti-MUC1 peptide mAb using MUC1 cDNA-transfected colon cancer CHC-Y1 cells as the target. The decreased sensitivity of MUC1 transfectants to effector cells was restored to a level equivalent to that in control cells. These data suggest that the detection of circulating anti-MUC1 antibodies may be a useful adjunct for the early diagnosis and immunological analysis of colorectal cancer.

Decreased expression of biliary glycoprotein in hepatocellular carcinomas

Biliary glycoprotein (BGP) is an adhesion and anti‐cell‐growth molecule of the carcinoembryonic antigen family. We have earlier demonstrated that BGP mRNA is expressed in hepatocellular carcinomas (HCCs) and the adjacent non‐cancerous regions, neither of which express CEA and NCA mRNA. To define an expression level and pattern of BGP at the protein level in HCCs, TS135, a monoclonal antibody (MAb) against BGP, was prepared. This MAb clearly reacted with BGP with a molecular weight of 110 kDa and 85 kDa (BGP‐110/85). It cross‐reacted weakly with NCA‐90 from NCA transfectants, but not at all with CEA‐200 from the serum of a colon‐cancer patient. The BGP transfectants of cultured hepatocellular carcinoma cHc‐4 cells showed Ca2−‐dependent cell aggregation, which was partially inhibited by modulating BGP on the cell surface with MAb TS135. Immunostaining of non‐cancerous liver tissues with MAb TS135 indicated that BGP could be expressed in the bile canalicular domain of hepatocytes. In HCCs, the expression of BGP was predominantly found in the well‐differentiated type, where the bile canaliculi and the apical portion of pseudoglands were positively stained, although their staining intensity and stained area were lower and more limited, respectively, than those of non‐cancerous regions. The percentage of faintly positive and negative cases (n = 22) from the total (n = 30) was 73%. This suggests that the expression level of BGP decreased in HCCs as compared with adjacent non‐cancerous regions. Int. J. Cancer 74:15–19.

Effect of MUC1 Mucin, an Anti‐adhesion Molecule, on Tumor Cell Growth

MUC1 mucin is expressed in a wide variety of tumors and is considered to function as an anti‐adhesion molecule which inhibits cell‐to‐cell interactions. To reveal the biological significance of this activity in tumor cells, MUC1 cDNA was transfected into EJNIH3T3 cells and human colon cancer cell lines, CHCY1 and DLD1. The in vivo growth rate of MUC1+ (MUC1‐transfected) EJNIH3T3, CHCY1 and DLD1 cells in SCID mice was clearly lower than that of MUC1− (mock transfectant) cells. Several in vitro experiments using MUC1+ EJNIH3T3 cells were performed to analyze the mechanisms for the decreased in vivo tumor growth. It was found that (i) the in vitro growth rate of MUC1+ EJNIH3T3 cells was also decreased compared to that of MUC1− cells, (ii) the DNA synthesis of MUC1+ EJNIH3T3 cells after stimulation with either growth factor (fetal calf serum or bombesin) or extracellular matrix (collagen or fibronectin) was lower than that of MUC1− cells, and (iii) MUC1+ EJNIH3T3 cells grew more slowly than MUC1− cells on both collagen‐ and fibronectin‐coated dishes. These data suggest that MUC1 mucin may regulate tumor cell growth through inhibition of cell‐to‐cell, growth factor‐to‐receptor and cell‐to‐matrix interactions.