Kohei Murata

Fucosylated haptoglobin is a novel marker for pancreatic cancer: A detailed analysis of the oligosaccharide structure and a possible mechanism for fucosylation

Changes in oligosaccharide structures have been reported in certain types of malignant transformations and, thus, could be used for tumor markers in certain types of cancer. In the case of pancreatic cancer cell lines, a variety of fucosylated proteins are secreted into their conditioned media. To identify fucosylated proteins in the serum of patients with pancreatic cancer, we performed western blot analyses using Aleuria Aurantica Lectin (AAL), which is specific for fucosylated structures. An ∼40 kD protein was found to be highly fucosylated in pancreatic cancer and an N‐terminal analysis revealed that it was the β chain of haptoglobin. While the appearance of fucosylated haptoglobin has been reported in other diseases such as hepatocellular carcinoma, liver cirrhosis, gastric cancer and colon cancer, the incidence was significantly higher in the case of pancreatic cancer. Fucosylated haptoglobin was observed more frequently at the advanced stage of pancreatic cancer and disappeared after an operation. A mass spectrometry analysis of haptoglobin purified from the serum of patients with pancreatic cancer and the medium from a pancreatic cancer cell line, PSN‐1, showed that the α 1‐3/α 1‐4/α 1‐6 fucosylation of haptoglobin was increased in pancreatic cancer. When a hepatoma cell line, Hep3B, was cultured with the conditioned media from pancreatic cancer cells, haptoglobin secretion was dramatically increased. These findings suggest that fucosylated haptoglobin could serve as a novel marker for pancreatic cancer. Two possibilities were considered in terms of the fucosylation of haptoglobin. One is that pancreatic cancer cells, themselves, produce fucosylated haptoglobin; the other is that pancreatic cancer produces a factor, which induces the production of fucosylated haptoglobin in the liver.

Genome-wide cDNA microarray analysis of gene expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelial cells selected for purity by laser microdissection

To characterize molecular mechanism involved in pancreatic carcinogenesis, we analysed gene-expression profiles of 18 pancreatic tumors using a cDNA microarray representing 23 040 genes. As pancreatic ductal adenocarcinomas usually contain a low proportion of cancer cells in the tumor mass, we prepared 95% pure populations of pancreatic cancer cells by means of laser microbeam microdissection, and compared their expression profiles to those of similarly purified, normal pancreatic ductal cells. We identified 260 genes that were commonly upregulated and 346 genes that were downregulated in pancreatic cancer cells. Because of the high degree of purity in the cell populations, a large proportion of genes that we detected as upregulated or downregulated in pancreatic cancers were different from those reported in previous studies. Comparison of clinicopathological parameters with the expression profiles indicated that altered expression of 76 genes was associated with lymph-node metastasis and that of 168 genes with liver metastasis. In addition, expression levels of 30 genes were related to the recurrence of disease. These genome-wide expression profiles should provide useful information for finding candidate genes whose products might serve as specific tumor markers and/or as molecular targets for treatment of patients with pancreatic cancer.

Prometastatic effect of N-acetylglucosaminyltransferase V is due to modification and stabilization of active matriptase by adding beta 1-6 GlcNAc branching.

Oligosaccharide moieties of glycoproteins are structurally altered during development, carcinogenesis, and malignant transformations. It is well known that β1–6 GlcNAc branching, a product of UDP-GlcNAc α-mannoside β1–6-N-acetylglucosaminyltransferase (GnT-V), is associated with malignant transformation as the results of such alterations. However, the mechanism by which β1–6 GlcNAc branching is linked to metastasis remains unclear, because the identification of specific glycoprotein(s) that are glycosylated by GnT-V and its biological function have not been examined. We herein report that matriptase, which activates both urokinase-type plasminogen activator and hepatocyte growth factor, is a target protein for GnT-V. The overexpression of GnT-V in gastric cancer cells leads to severe peritoneal dissemination in athymic mice, which can be attributed to the increased expression of matriptase. This increase was due to the acquired resistance of matriptase to degradation, since it is glycosylated by GnT-V and a corresponding increase in the active form. These results indicate that this process is a key element in malignant transformation, as the direct result of oligosaccharide modification.

Expression of S100A6 and S100A4 in Matched Samples of Human Colorectal Mucosa, Primary Colorectal Adenocarcinomas and Liver Metastases

Objective: S100A6 and S100A4, two of S100 protein family, have been suggested to be associated with cancer tumorigenesis and metastasis. The aim of this study was to evaluate the expression levels of S100A6 and S100A4 in matched samples of primary human colorectal adenocarcinomas (T), adjacent normal colorectal mucosa (N) and liver metastases (M). This gave us the advantage of directly comparing levels of S100A6 and S100A4 expression within the same genetic background. Methods: In matched samples of N, T and M from 10 colorectal adenocarcinoma patients, expressions of S100A6 and S100A4 were studied by Western blot and immunohistochemical analyses using specific antibodies against each protein. Results: The expression levels of S100A6 were significantly higher in T than in N (p < 0.05), while those of S100A4 showed no difference between T and N. There were no significant differences in the expression levels of S100A6 or S100A4 between M and T. Similar results were obtained by immunohistochemical analysis. Moreover, S100A6 was stained more intensely in invading fronts than in central portions of both T and M. Conclusions: The observed differential expression of S100A6 and S100A4 suggests that S100A6, rather than S100A4, is associated with human colorectal adenocarcinoma tumorigenesis and invasion/metastasis.

Surgery for pulmonary metastases from colorectal cancer: the importance of prethoracotomy serum carcinoembryonic antigen as an indicator of prognosis.

OBJECTIVE Several investigators have analyzed prognostic factors of surgical treatment for pulmonary metastases from colorectal cancer, but the results remain inconclusive. This study was performed to determine the prognostic implications of the prethoracotomy serum level of carcinoembryonic antigen (CEA) in relation to the postthoracotomy recurrent pattern among patients with this disease. METHODS A retrospective analysis of prognostic factors was undertaken in 100 patients who had consecutively undergone initial surgical resection for pulmonary metastases of colorectal origin. RESULTS The overall 3- and 5-year survival rates were 62.2% and 49.4%, respectively. Univariate analysis revealed that the prethoracotomy serum CEA level and operative curability were strongly associated with prognosis, while in multivariate analysis, only the prethoracotomy serum CEA level was a significant prognostic indicator. Patients with a high level of prethoracotomy serum CEA more frequently exhibited recurrence in extrathoracic sites, especially in the brain. CONCLUSION Before thoracotomy for pulmonary metastases from colorectal cancer, the serum CEA level was the most useful prognostic factor. Patients with elevated serum CEA level should undergo a careful prethoracotomy systemic survey and postthoracotomy follow-up for extrathoracic metastases, in particular brain metastases, and an appropriate combined therapeutic modality should be considered.

Aberrant Expression of Connexin 26 Is Associated with Lung Metastasis of Colorectal Cancer

Purpose: Connexin 26 (Cx26) is one of the gap junction–forming family members classically considered to be tumor suppressors. However, recent studies show association of elevated expression of Cx26 with poor prognosis in several human malignancies. Furthermore, Cx26 has been observed to be indispensable to spontaneous metastasis of melanoma cells. Here, we assessed Cx26 expression in primary colorectal cancer (CRC) and the metastatic lesions to elucidate its role in metastasis. Experimental Design: Cx26 expression was assessed in 25 adenomas, 167 CRCs, and normal mucosa, together with the metastatic lesions. Results: Normal mucosa and adenomatous tissue expressed Cx26 mainly in the plasma membrane, whereas cancer cells mostly contained Cx26 in the cytoplasm. The incidence of aberrant Cx26 expression varied widely in CRC (mean, 49.5 ± 35.5%), and the expression levels were confirmed by Western blot and quantitative reverse transcription–PCR. Clinicopathologic survey revealed association of high expression with less differentiated histology and venous invasion (P = 0.0053 and P = 0.0084, respectively). Notably, high Cx26 expression was associated with shorter disease-free survival and shorter lung metastasis–free survival in 154 curatively resected CRC sets (P = 0.041 and P = 0.028, respectively). Survey of metastatic lesions revealed that lung metastasis, but not liver and lymph nodes metastases, expressed higher Cx26 than the CRC series or corresponding primary CRCs (P < 0.0001 and P = 0.0001, respectively). Conclusions: These findings suggest that aberrant expression of Cx26 plays an essential role in lung metastasis. Thus, Cx26 is a promising therapeutic target, particularly for CRC patients who develop lung metastasis.

Fucosylated haptoglobin is a novel type of cancer biomarker linked to the prognosis after an operation in colorectal cancer.

Fucosylation is a crucial oligosaccharide modification in cancer and inflammation. Total cellular proteins of cancer cells and the sera of patients with cancer both show increased fucosylation levels. Certain kinds of fucosylated proteins can be applied as novel cancer biomarkers in glyco‐proteomic analyses. We previously identified fucosylated haptoglobin (Fuc‐Hpt) as a serologic marker for pancreatic cancer, and recently developed a lectin‐antibody enzyme‐linked immunosorbent assay system for quantifying Fuc‐Hpt. In the present study, we investigated the clinical outcome of Fuc‐Hpt levels in patients with colorectal cancer (CRC), and examined the mechanisms underlying Fut‐Hpt production using a murine tumor transplantation model.

Association of the Primary Tumor Location with the Site of Tumor Recurrence after Curative Resection of Thoracic Esophageal Carcinoma

The site of surgical failure in cases of thoracic esophageal cancer (TEC) may be affected by the vertical location of the cancer in this longitudinal organ, suggesting the need to select the mode of adjuvant therapy based on location. We classified 501 TECs (92% squamous cell carcinomas) that underwent curative surgery without preoperative treatment as 13% upper thoracic (Ut), 51% middle thoracic (Mt), and 36% lower thoracic (Lt) lesions. Recurrent disease was discovered in 180 (36%) of the patients during a postoperative survey, most frequently in the cervical nodes (19%), liver (18%), abdominal paraaortic nodes (17%), and upper mediastinal nodes (17%). Although postoperative survival rates were similar (5-year survival: Ut 51%, Mt 55%, Lt 54%), the tumor recurrence site was significantly affected by the TEC vertical location, with recurrence in the cervical and upper mediastinal nodes being most frequent for Ut and Mt cases and in the liver and abdominal paraaortic nodes for Lt cases. Insufficient surgical lymph node clearance could be assessed by the recurrence index (RI), defined as the frequency of metastasis at recurrence divided by that at surgery. The RI was significantly lower for the upper abdominal nodes (4%, 8/184) than the lower mediastinal nodes (15%, 19/123) or the upper mediastinal nodes (19%, 30/154). These findings indicated that regional tumor recurrence, corresponding to the surgical field, was more frequent in the Ut and Mt cases (53% and 51%) than the Lt cases (18%); and distant recurrence was more frequent in the Lt cases (62%) than in Ut or Mt cases (25% and 36%). Thus the vertical location of the thoracic esophageal cancer can be said to affected strongly the site of tumor recurrence after curative surgery. Regional radiotherapy might be expected to have an adjuvant effect on Ut/Mt tumors and systemic chemotherapy on Lt tumors.

Decreased miR-340 expression in bone marrow is associated with liver metastasis of colorectal cancer

Studies have shown the prognostic significance of disseminated tumor cells (DTC) in bone marrow of patients with colorectal cancer. However, the molecular characteristics of DTCs, including their miRNA expression profiles, remain mostly unknown. In this study, we analyzed the miRNA expression of DTCs in bone marrow. EpCAM+ bone marrow cells were collected using immunomagnetic beads after exclusion of CD14+ and CD45+ cells, then subjected to miRNA microarray analysis. Cluster analysis (7 colorectal cancer patients with liver metastasis and 12 colorectal cancer patients without liver metastasis) indicated that miR-340 and miR-542-3p expressions were significantly decreased in EpCAM+ bone marrow cells of patients with liver metastasis (P = 0.019 and 0.037, respectively). We demonstrated that pre-miR-340 administration inhibited growth of colon cancer cells and suppressed c-Met expression in vitro. In clinical samples of colorectal cancer, miR-340 was expressed at significantly lower levels in tumor tissues compared with normal mucosa. Survival analysis in 136 patients with colorectal cancer indicated that low miR-340 expression was correlated with shorter 5-year disease-free survival (P = 0.023) and poor 5-year overall survival (P = 0.046). It was of note that the colorectal cancer group with low miR-340 and high c-Met expression had the worst prognosis. We further demonstrated that systemic pre-miR-340 administration suppressed growth of pre-established HCT116 tumors in animal therapeutic models. These findings indicate that miR-340 may be useful as a novel prognostic factor and as a therapeutic tool against colorectal cancer. Our data suggest that miR-340 in bone marrow may play an important role in regulating the metastasis cascade of colorectal cancer. Mol Cancer Ther; 13(4); 976–85. ©2014 AACR.

Activation of Insulin-like Growth Factor Signaling Induces Apoptotic Cell Death Under Prolonged Hypoxia by Enhancing Endoplasmic Reticulum Stress Response

Malignant cells in solid tumors survive under prolonged hypoxia and can be a source of resistance to current cancer therapies. Mammalian target of rapamycin (mTOR), one of the downstream molecules of the insulin-like growth factor (IGF) pathway, is a key regulator of translation, integrating multiple environmental and nutritional cues. The activity of mTOR is known to be suppressed under hypoxic conditions in cancer cells, whereas the contribution of this suppression to cell survival has not yet been clarified. We show that stimulating IGF signaling provoked caspase-dependent apoptosis under low oxygen tension in two cancer cell lines, COLO 320 and AsPC-1. In concurrence with increased levels of BAD phosphorylation, cell death was not accompanied by cytochrome c release from mitochondria. The cells were rescued from apoptosis when phosphatidylinositol 3-kinase (PI3K) or mTOR activity was inhibited, suggesting that these signals are critical in the observed cell death. IGFs and insulin enhanced the endoplasmic reticulum (ER) stress response as monitored by induction of the CCAAT/enhancer binding protein homologous protein (CHOP) proteins and the X box protein-1 splicing under hypoxic conditions, and this response was suppressed by inhibiting PI3K and mTOR activity. IGF-induced cell death under hypoxic conditions was prevented by treatment with cycloheximide, suggesting that de novo protein synthesis is required. Indeed, suppression of CHOP protein levels with small hairpin RNA reduced cell death. Taken together, the data suggest that stimulating IGF signaling under hypoxic conditions provokes apoptosis by enhancing the ER stress response.