Nobuyoshi Momiyama

Matrilysin-specific antisense oligonucleotide inhibits liver metastasis of human colon cancer cells in a nude mouse model

Human colon cancer frequently develops liver metastasis. Matrilysin (MMP‐7), the smallest member of the matrix metalloproteinase (MMP) family, is commonly produced by human colon carcinoma cells and has been suggested to be involved in the progression and metastasis of this type of cancer. In the present study, we tested the effect of a matrilysin‐specific antisense phosphorothioate oligonucleotide on liver metastasis of the human colon carcinoma cell line WiDr in nude mice. In culture, the antisense oligonucleotide moderately inhibited the secretion of matrilysin by WiDr cells. Injection of WiDr cells into the spleen of nude mice produced many metastatic tumor nodules in the liver. When the antisense oligonucleotide was injected daily into the mice for 11 days, the formation of the metastatic tumor nodules was strongly inhibited in a dose‐dependent manner. An inhibition of liver metastasis of over 70% was obtained at a dose of 120 μg of the oligonucleotide per mouse. The antisense oligonucleotide did not inhibit tumor growth in spleen and in liver. A scrambled control oligonucleotide had no effect on liver metastasis of WiDr cells. Our results demonstrate an important role of matrilysin in liver metastasis of human colon cancer and the therapeutic potential of matrilysin antisense oligonucleotides for the prevention of metastasis. Int. J. Cancer 76:812–816, 1998.© 1998 Wiley‐Liss, Inc.

Rapid Detection of Epidermal Growth Factor Receptor Mutations in Lung Cancer by the SMart-Amplification Process

Purpose: A positive response to gefitinib in non–small cell lung cancer (NSCLC) has been correlated to mutations in epidermal growth factor receptor (EGFR) gene. Previous reports have been based mainly on diagnostic screening by sequencing. However, sequencing is a time-consuming and complicated procedure, not suitable for routine clinical use. Experimental Design: We have developed rapid, simple, and sensitive mutation detection assays based on the SMart Amplification Process (SMAP) and applied it for analyzing EGFR gene mutations in clinical samples. By using SMAP, we can detect mutations within 30 min including sample preparation. To validate the assay system for potential use in clinical diagnostics, we examined 45 NSCLC patients for EGFR mutations using sequencing and SMAP. Results: The outcomes of the SMAP assay perfectly matched the sequencing results, except in one case where SMAP was able to identify a mutation that was not detected by sequencing. We also evaluated the sensitivity and specificity of SMAP in mutation detection for EGFR. In a serial dilution study, SMAP was able to find a mutation in a sample containing only 0.1% of the mutant allele in a mixture of wild-type genomic DNA. We also could show amplification of mutated DNA with only 30 copies per reaction. Conclusions: The SMAP method offers higher sensitivity and specificity than alternative technologies, while eliminating the need for sequencing to identify mutations in the EGFR gene of NSCLC. It provides a robust and point-of-care accessible approach for a rapid identification of most patients likely to respond to gefitinib.

Matrilysin is associated with progression of colorectal tumor

Matrilysin and gelatinase A, B mRNA expressions were examined in colorectal tumors. Matrilysin mRNA was observed exclusively in tumors, while the others were also found in normal mucosa surrounding tumors. Further analysis revealed that colorectal adenomas with severe dysplasia, not with mild dysplasia, expressed matrilysin with lower levels than cancers. The level of matrilysin mRNA expression increased with the advancement of stages of colorectal cancers, consequently a relatively higher expression was observed in liver metastatic tumors than primary tumors. These results suggest that matrilysin mRNA expression was correlated with the progression of colorectal tumors, and this enzyme may also play a role in developing metastatic tumors in liver.

Predicting 5-fluorouracil chemosensitivity of liver metastases from colorectal cancer using primary tumor specimens: Three-gene expression model predicts clinical response

We identified genes related to 5‐fluorouracil (5‐FU) sensitivity in colorectal cancer and utilized these genes for predicting the 5‐FU sensitivity of liver metastases. Eighty‐one candidate genes involved in 5‐FU resistance in gastric and colon cancer cell lines were previously identified using a cDNA microarray. In this study, the mRNA expression levels of these 81 selected genes and the genes of 5‐FU‐related enzymes, including thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyltransferase (OPRT), were measured using real‐time quantitative RT‐PCR assays of surgically resected materials from primary colorectal tumors in 22 patients. Clinical responses were estimated by evaluating the effects of 5‐FU‐based hepatic artery injection (HAI) chemotherapy for synchronous liver metastases. Four genes (TNFRSF1B, SLC35F5, NAG‐1 and OPRT) had significantly different expression profiles in 5‐FU‐nonresponding and responding tumors (p < 0.05). A “Response Index” system using three genes (TNFRSF1B, SLC35F5 and OPRT) was then developed using a discriminate analysis; the results were well correlated with the individual chemosensitivities. Among the 11 cases with positive scores in our response index, 9 achieved a reduction in their liver metastases after 5‐FU‐based chemotherapy, whereas only 1 of the 11 cases with negative scores responded well to chemotherapy. Our “Response Index” system, consisting of TNFRSF1B, SLC35F5 and OPRT, has great potential for predicting the efficacy of 5‐FU‐based chemotherapy against liver metastases from colorectal cancer.

Matrilysin stimulates DNA synthesis of cultured vascular endothelial cells and induces angiogenesis in vivo.

Matrilysin produced by human colon cancer cells may be involved in the progression and metastasis of cancer. In the present study, we investigated the association of matrilysin with angiogenesis. One microgram of recombinant matrilysin is confirmed to have increased [3H]-thymidine uptake in human umbilical vein endothelial cells. Then we used micro encapsulation and a mouse hemoglobin enzyme-linked immunosorbent assay system for in vivo quantitation of angiogenesis with BALB/c nu/nu athymic mice. Hundred micrograms of recombinant matrilysin induced angiogenesis to the same degree as 10 microg of basic fibroblast growth factor (bFGF). Angiogenesis was observed at the site implanted with human colon cancer WiDr cells in agarose micro beads. This was inhibited by subcutaneous injection of matrilysin-specific antisense oligonucleotide significantly by 53%. In conclusion, matrilysin may be associated with angiogenesis of human colon cancer through the direct proliferative action on endothelial cells.

Expression of highly polysialylated neural cell adhesion molecule in pancreatic cancer neural invasive lesion

Neurotropism of pancreatic cancer is one of the hypotheses explaining neural invasion, which is one of the characteristics of pancreatic cancer. In these studies, we immunohistochemically examined neural cell adhesion molecules (NCAM), homophilic adhesion molecules expressed on the nerve cells, as a factor of neurotropism, in 15 pancreatic cancer operatively obtained, especially in neural invasive lesions. We also investigated the role of polysialic acid (PSA), which is attached to NCAM and related to the malignant potential of cancers. NCAM was detected in 66.7% of pancreatic cancers, and in all 9 cases with massive perineural invasion. In neural invasive lesions, however, there were perineurium and endoneurium, which do not express NCAM, between the cancer and nerve cells. PSA was also detected in the pancreatic cancers expressing NCAM. Moreover, PSA expression was stronger in the perineural invasive lesions than in the main tumor and was related to the cancer cell proliferation investigated by Ki-67 staining. It is unlikely therefore, that NCAM plays an important role in neurotropism. However, the NCAM expressed on the pancreatic cancer was attached to PSA, which itself plays an important role in the malignant potential of this disease.

DETECTION OF REGIONAL LYMPH NODE METASTASES IN COLON CANCER BY USING RT-PCR FOR MATRIX METALLOPROTEINASE 7, MATRILYSIN

Lymph node metastasis is the most important prognostic factor in colon cancer. However, more accurate screening for metastasis than that afforded by conventional pathology remains elusive. We have employed a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for a matrix metalloproteinase (MMP), ‘matrilysin’, because this gene is epithelial-specific and consistently expressed in colorectal cancer cells. The sensitivity of this assay was examined with the matrilysin-producing rectal cancer cell line ‘CaR-1’. Matrilysin mRNA was detected in this system when more than 10 matrilysin-positive cells existed in a lymph node of ordinary size. Fourteen of 15 (93%) primary colon cancers and none of the surrounding normal tissues expressed matrilysin. All 10 histologically-positive lymph nodes were positive for matrilysin, while of 60 histologically-negative lymph nodes, eight were positive for matrilysin. When the additional sequential sectioning and histological re-examinati on was performed on five of these eight ‘matrilysin-positive, but histologically-negative’ lymph nodes, micrometastases were detected in three. Only one of the lymph nodes that were histologically-positive, but negative by matrilysin assay was from a patient with colon cancer in which matrilysin was not detected. In conclusion, RT-PCR assay for matrilysin is a sensitive method for detecting occult metastases in patients with colon cancer, and may complement histologic examination.

Matrilysin gene expression in sporadic and familial colorectal adenomas

We examined the expression of matrilysin mRNA in sporadic and hereditary colorectal adenomas to clarify the role of matrilysin in tumorigenesis. Matrilysin mRNA was not detected in normal colorectal mucosa from patients with either sporadic or familial adenomas. Matrilysin mRNA expression in sporadic adenomas correlated with the degree of dysplasia and the size of the mass, whereas most of the adenomas in patients with familial adenomatous polyposis coli expressed matrilysin mRNA irrespective of adenoma size or degree of dysplasia. Because matrilysin is more likely to be expressed in adenomas with a potential for malignancy, this enzyme may play a role in the malignant conversion of colorectal adenomas. Mol. Carcinog. 19:225–229, 1997.

Inhibition of osteoclast differentiation and bone resorption by cathepsin K antisense oligonucleotides

We confirmed the expression of cathepsin K, the most abundant and specific cysteine protease found in osteoclasts, at the mRNA level in most of our cases of breast cancer, and even at the protein level in bone metastatic lesions. Therefore, we investigated the functions of cathepsin K in osteoclasts with special attention to bone metastasis from breast cancer. Mouse osteoclast‐like cells (OCLs) were established by coculture of mouse bone marrow cells and osteoblastic cells. Rodent cathepsin K antisense (AS) or random control (CL) oligonucleotides were added on day 0, 3, or 6 of culture. Tartrate‐resistant acid phosphatase staining confirmed the formation of OCLs after 9 d of incubation. AS treatment significantly reduced both the number of TRAP‐positive cells and the percentage of multinuclear cells. For the pit‐forming assay, after 9 d of incubation, mature OCLs were collected and incubated on ivory slices with AS or CL for 48 h. The antisense oligonucleotides also inhibited the bone‐resorbing activity of OCLs. CL treatment did not affect either the number of TRAP‐positive cells or pit formation. Cathepsin K may play important roles in bone resorption as well as in differentiation of osteoclasts. These findings indicate that the inhibition of this enzyme may prevent the development of bone metastasis from breast cancer.

Amphiregulin Is a Promising Prognostic Marker for Liver Metastases of Colorectal Cancer

Purpose: Aberrant activation of epidermal growth factor receptors (EGFR/HER1) by ligand stimulation or heterodimerization with human epidermal growth factor 2 (HER2) is considered to play an important role in the development of colorectal carcinoma. Amphiregulin (AR) is a ligand of EGFR that might be related to the development and progression of gastrointestinal tumors. The aim of this study was to determine the AR, EGFR, and HER2 protein expression levels and to evaluate their prognostic relevance to the clinical course of colorectal cancer. Experimental Design: The AR, EGFR, and HER2 protein levels in primary tumors of colorectal cancer (n = 106) were examined using immunohistochemistry. Metastatic sites in liver specimens (n = 16) were also analyzed in the same manner. Results: Thirteen (81.6%) metastatic lesions of the liver stained positive for AR. Among the primary lesions of colorectal cancer, 58 (54.7%) stained positive for AR, 13 (12.3%) stained positive for EGFR, and 5 (4.7%) stained positive for HER2. When the relationships between each protein expression level and the clinicopathologic factors were examined, only the AR expression level was significantly related to liver metastasis (P = 0.0296). A multivariate analysis of liver metastasis proved that AR expression was an independent prognostic factor of liver metastasis from colorectal cancer (P = 0.0217). Conclusions: AR expression in primary lesions of colorectal cancer is an important predictive marker of liver metastasis.