Shyuichiro Matsubara

A new family of heparin-binding factors: strong conservation of midkine (MK) sequences between the human and the mouse.

A retinoic acid responsive gene, MK, specifies for a heparin binding factor termed midkine (MK), which is the initial member of a new protein family involved in regulation of growth and differentiation. A cDNA clone of human MK was isolated from a fetal kidney cDNA library. Human MK mRNA was expressed in PA1 teratocarcinoma cells as well as in the kidney. Sequence analysis of the cDNA clone and of a part of the genomic clone yielded the predicted protein sequence of human MK. Human and mouse MK sequences are highly conserved: 87% of amino acids are identical and all amino acid changes are conservative except for an insertion. Comparison of MK and HB-GAM/pleiotrophin (another member of the family) from various species revealed sequences conserved in the family and those specific for each protein.

mTOR plays critical roles in pancreatic cancer stem cells through specific and stemness-related functions

Pancreatic cancer is characterized by near-universal mutations in KRAS. The mammalian target of rapamycin (mTOR), which functions downstream of RAS, has divergent effects on stem cells. In the present study, we investigated the significance of the mTOR pathway in maintaining the properties of pancreatic cancer stem cells. The mTOR inhibitor, rapamycin, reduced the viability of CD133+ pancreatic cancer cells and sphere formation which is an index of self-renewal of stem-like cells, indicating that the mTOR pathway functions to maintain cancer stem-like cells. Further, rapamycin had different effects on CD133+ cells compared to cyclopamine which is an inhibitor of the Hedgehog pathway. Thus, the mTOR pathway has a distinct role although both pathways maintain pancreatic cancer stem cells. Therefore, mTOR might be a promising target to eliminate pancreatic cancer stem cells.

Establishment of a highly migratory subclone reveals that CD133 contributes to migration and invasion through epithelial–mesenchymal transition in pancreatic cancer

Pancreatic cancer is a lethal disease because of invasion and early metastasis. Although CD133, a marker of cancer stem cells (CSCs) in a variety of solid tumors, has been studied in recent decades, its function remains obscure. Recent reports suggest that epithelial–mesenchymal transition (EMT) may be related to the properties of CSCs. In this study, we investigated whether CSC markers are associated with EMT. For Capan1M9, a highly migratory cell subclone established from human pancreatic cancer cell line Capan-1, CD133 expression, migration, and invasion were greater than for the parent cells. In Capan1M9 cells, the EMT-related transcription factors Slug and Snail were up-regulated, and N-cadherin and fibronectin were also substantially increased. In contrast, occludin and desmoplakin were suppressed. Knockdown of endogenous CD133 in the Capan1M9 cells led to Slug suppression and reduction of migration and invasion. Taken together, CD133 has an important role in migration and invasion by facilitating EMT in pancreatic cancer cells.

Chromosomal localization of two cell surface-associated molecules of potential importance in development: midkine (Mdk) and basigin (Bsg).

Midkine (protein symbol: MK, proposed gene symbol: Mdk) is the first member of a family of heparin binding growth factors (Kadomatsu et al. 1988; Tomomura et al. 1990a, 1990b). MK has mitogen activity on PC12 pheochromocytoma cells (Tomomura et al. 1990b), while heparin binding growth associated molecule (HB-GAM), another member of the family, has neurite extension activity and is mitogenic for fibroblasts (Merenmies and Rauvala 1990; Li et al. 1990). MK has a molecular weight of 14,000 and is rich both in basic amino acids and cysteine (Tomomura et al. t990a). MK and HB-GAM have 50% homology and all cysteines are conserved (Merenmies and Rauvala 1990; Li et al. 1990; Tsutsui et al. 1991). Since MK has a strong affinity for heparin it should be mainly localized at the surface of the cell either in a matrix-bound form or in a membrane-bound form. Mdk, the gene coding for MK, is a retinoic acid responsive gene and it is expressed during the mid-gestation period of mouse embryogenesis (Kadomatsu et al. 1988, 1990; Huang et al. 1990). In adult mice, only the kidney expresses Mdk significantly (Kadomatsu et al. 1988). Basigin (gene symbol: Bsg) is a member of the immunoglobulin superfamily, and displays strong homology with both immunoglobulin V domain and the C-domain-like sequence of major histocompatibility antigen class II 13 chain (Miyaushi et al. 1990). The immunoglobulin-like domain of basigin may be closely related to the primordial form of the immunoglobulin domains. As other molecules of the immunoglobulin superfamily, basigin is expected to be involved in cell surface interactions as an adhesion molecule or as a receptor (Williams and Barclay 1988). Although basigin was found as a molecule bearing a carbohydrate marker of embryonic nature, the gene coding for bas-

Expression of α-catenin in α-catenin–deficient cells increases resistance to sphingosine-induced apoptosis

α-Catenin, an intracellular protein, associates with the COOH-terminal region of cadherin cell adhesion molecules through interactions with either β-catenin or γ-catenin (plakoglobin). The full activity of cadherins requires a linkage to the actin cytoskeleton mediated by catenins. We transfected α-catenin–deficient colon carcinoma cells with a series of α-catenin constructs to determine that α-catenin expression increases the resistance to apoptosis induced by sphingosine. Two groups of constructs, containing deletions in either the middle segment of the molecule or the COOH terminus, induced morphological changes, cell compaction, and decreases in cell death. In α-catenin–expressing cells, inhibition of cadherin cell adhesion by treatment with anti–E-cadherin antibodies did not decrease the cells viability. α-Catenin expression partially suppressed the downregulation of Bcl-xL and the activation of caspase 3. Expression of p27kip1 protein, an inhibitor of cyclin-dependent kinases, was increased by α-catenin expression in low density cell cultures. The increased levels of p27kip1 correlated with both increased resistance to cell death and morphological changes in transfectants containing deletion mutants. Transfection-mediated upregulation of p27kip1 decreases sphingosine-induced cell death in α-catenin–deficient cells. We postulate that α-catenin mediates transduction of signals from the cadherin–catenin complex to regulate the apoptotic cascade via p27kip1.

CD133 facilitates epithelial-mesenchymal transition through interaction with the ERK pathway in pancreatic cancer metastasis

BackgroundPancreatic cancer is a lethal disease due to the high incidence of metastasis at the time of detection. CD133 expression in clinical pancreatic cancer correlates with poor prognosis and metastasis. However, the molecular mechanism of CD133-regulated metastasis remains unclear. In recent years, epithelial-mesenchymal transition (EMT) has been linked to cancer invasion and metastasis. In the present study we investigated the role of CD133 in pancreatic cancer metastasis and its potential regulatory network.MethodsA highly migratory pancreatic cancer cell line, Capan1M9, was established previously. After shRNA was stable transducted to knock down CD133 in Capan1M9 cells, gene expression was profiled by DNA microarray. Orthotopic, splenic and intravenous transplantation mouse models were set up to examine the tumorigenesis and metastatic capabilities of these cells. In further experiments, real-time RT-PCR, Western blot and co-immunoprecipitate were conducted to evaluate the interactions of CD133, Slug, N-cadherin, ERK1/2 and SRC.ResultsWe found that CD133+ human pancreatic cancer cells were prone to generating metastatic nodules in in vivo models using immunodeficient mice. In contrast, CD133 knockdown suppressed cancer invasion and metastasis in vivo. Gene profiling analysis suggested that CD133 modulated mesenchymal characteristics including the expression of EMT-related genes, such as Slug and N-cadherin. These genes were down-regulated following CD133 knockdown. Moreover, CD133 expression could be modulated by the extracellular signal-regulated kinase (ERK)1/2 and SRC signaling pathways. The binding of CD133 to ERK1/2 and SRC acts as an indispensable mediator of N-cadherin expression.ConclusionsThese results demonstrate that CD133 plays a critical role in facilitating the EMT regulatory loop, specifically by upregulating N-cadherin expression, leading to the invasion and metastasis of pancreatic cancer cells. Our study provides a novel insight into the function of CD133 in the EMT program and a better understanding of the mechanism underlying the involvement of CD133 in pancreatic cancer metastasis.

miR-30 family promotes migratory and invasive abilities in CD133(+) pancreatic cancer stem-like cells.

Pancreatic cancer is a deadly disease with a poor prognosis. Recently, miRNAs have been reported to be abnormally expressed in several cancers and play a role in cancer development and progression. However, the role of miRNA in cancer stem cells remains unclear. Therefore, our aim was to investigate the role of miRNA in the CD133+ pancreatic cancer cell line Capan-1M9 because CD133 is a putative marker of pancreatic cancer stem cells. Using miRNA microarray, we found that the expression level of the miR-30 family decreased in CD133 genetic knockdown shCD133 Capan-1M9 cells. We focused on miR-30a, -30b, and -30c in the miR-30 family and created pancreatic cancer cell sublines, each transfected with these miRNAs. High expression of miR-30a, -30b, or -30c had no effect on cell proliferation and sphere forming. In contrast, these sublines were resistant to gemcitabine, which is a standard anticancer drug for pancreatic cancer, and in addition, promoted migration and invasion. Moreover, mesenchymal markers were up-regulated by these miRNAs, suggesting that mesenchymal phenotype is associated with an increase in migration and invasion. Thus, our study demonstrated that high expression of the miR-30 family modulated by CD133 promotes migratory and invasive abilities in CD133+ pancreatic cancer cells. These findings suggest that targeted therapies to the miR-30 family contribute to the development of novel therapies for CD133+ pancreatic cancer stem cells.

Expression of |[alpha]|-catenin in |[alpha]|-catenin-deficient cells results in a reduced proliferation in three-dimensional multicellular spheroids but not in two-dimensional monolayer cultures

α-Catenin is an intracellular protein that associates with the carboxy-terminal region of cadherin, a cell adhesion molecule, via β-catenin or γ-catenin (plakoglobin). Linkage of cadherin to the cytoskeleton by catenins is required for full cadherin activity. Following transfection of an α-catenin-deficient colon carcinoma cell line with a series of α-catenin constructs, we discovered that the restoration of α-catenin expression results in reduced proliferation in three-dimensional multicellular spheroids, but not in two-dimensional monolayer cultures. The cellular function of α-catenin has not been compared between cells in three- and two-dimensional culture; this is the first evidence that growth regulation in three-dimensional cultures requires signaling mediated by α-catenin. Two classes of constructs, containing deletions in either the central segment or the COOH terminus of the molecule, both induced morphological changes, including cell compaction, and suppressed cell growth in three-dimensional cultures. In α-catenin-expressing cells, inhibition of cadherin cell adhesion by treatment with anti-E-cadherin antibodies resulted in a similar phenotype as that observed following the loss of α-catenin. Therefore, both the homophilic interaction of the cadherin extracellular domain and the linkage of the cadherin cytoplasmic domain to the actin cytoskeleton by α-catenin are necessary for growth control in three-dimensional culture.

Expression of MUC17 Is Regulated by HIF1α-Mediated Hypoxic Responses and Requires a Methylation-Free Hypoxia Responsible Element in Pancreatic Cancer

MUC17 is a type 1 membrane-bound glycoprotein that is mainly expressed in the digestive tract. Recent studies have demonstrated that the aberrant overexpression of MUC17 is correlated with the malignant potential of pancreatic ductal adenocarcinomas (PDACs); however, the exact regulatory mechanism of MUC17 expression has yet to be identified. Here, we provide the first report of the MUC17 regulatory mechanism under hypoxia, an essential feature of the tumor microenvironment and a driving force of cancer progression. Our data revealed that MUC17 was significantly induced by hypoxic stimulation through a hypoxia-inducible factor 1α (HIF1α)-dependent pathway in some pancreatic cancer cells (e.g., AsPC1), whereas other pancreatic cancer cells (e.g., BxPC3) exhibited little response to hypoxia. Interestingly, these low-responsive cells have highly methylated CpG motifs within the hypoxia responsive element (HRE, 5′-RCGTG-3′), a binding site for HIF1α. Thus, we investigated the demethylation effects of CpG at HRE on the hypoxic induction of MUC17. Treatment of low-responsive cells with 5-aza-2′-deoxycytidine followed by additional hypoxic incubation resulted in the restoration of hypoxic MUC17 induction. Furthermore, DNA methylation of HRE in pancreatic tissues from patients with PDACs showed higher hypomethylation status as compared to those from non-cancerous tissues, and hypomethylation was also correlated with MUC17 mRNA expression. Taken together, these findings suggested that the HIF1α-mediated hypoxic signal pathway contributes to MUC17 expression, and DNA methylation of HRE could be a determinant of the hypoxic inducibility of MUC17 in pancreatic cancer cells.

Slug contributes to gemcitabine resistance through epithelial-mesenchymal transition in CD133+ pancreatic cancer cells

CD133-positive pancreatic cancer is correlated with unfavorable survival despite current development of therapy. Slug acts as a master regulator of epithelial-mesenchymal transition (EMT) which is the essential process in cancer progression. The aim of this study was to investigate the role of Slug in gemcitabine treatment for CD133-positive pancreatic cancer cells. We used a previously established pancreatic cancer cell line expressing high level of CD133 (Capan-1M9), which also expresses high level of Slug. We generated Slug knock-down subclone (shSlug M9) from this cell line, and compared expression of EMT-related genes, migration, invasion and gemcitabine resistance between two cell lines. Slug knock-down in CD133-positive pancreatic cancer cell line led to the reduction of migration and invasion ability. Furthermore, Slug knock-down sensitized CD133-positive pancreatic cancer cell line to gemcitabine. These results suggest that Slug plays an important role in not only invasion ability through EMT but also gemcitabine resistance of CD133-positive pancreatic cancer cells.