Haruhisa Maruyama

The TGF-β signaling inhibitor Smad7 enhances tumorigenicity in pancreatic cancer

Transforming growth factor-beta (TGF-β) signaling is dependent on the heterodimerization of the type II TGF-β receptor (TβRII) with the type I TGF-β receptor (TβRI). Activated TβRI then mediates TGF-β signals by inducing the phosphorylation of Smad2 and/or Smad3, which separately hetetorodimerize with Smad4 and translocate to the nucleus. Phosphorylation of Smad2/Smad3 by activated TβRI is inhibited by two newly discovered members of the Smad family, Smad6 and Smad7. We now report that Smad7 mRNA levels are increased in human pancreatic cancer by comparison with the normal pancreas, and that by in situ hybridization, Smad7 is over-expressed in the cancer cells within the tumor mass. Stable transfection of COLO-357 human pancreatic cancer cells with a full-length Smad7 construct leads to complete loss of the growth inhibitory response to TGF-β1, without altering TGF-β1-mediated induction of PAI-I. Furthermore, Smad7 transfected COLO-357 cells display enhanced anchorage-independent growth and accelerated growth in nude mice. These findings point to a previously unrecognized mechanism for selective suppression of TGF-β-mediated growth inhibition in cancer cells that allows for continued activation of the PAI-I promoter by TGF-β1, which may act to enhance the tumorigenicity of certain cancer cells.

Id-1 and Id-2 Are Overexpressed in Pancreatic Cancer and in Dysplastic Lesions in Chronic Pancreatitis

Id proteins antagonize basic helix-loop-helix proteins, inhibit differentiation, and enhance cell proliferation. In this study we compared the expression of Id-1, Id-2, and Id-3 in the normal pancreas, in pancreatic cancer, and in chronic pancreatitis (CP). Northern blot analysis demonstrated that all three Id mRNA species were expressed at high levels in pancreatic cancer samples by comparison with normal or CP samples. Pancreatic cancer cell lines frequently coexpressed all three Ids, exhibiting a good correlation between Id mRNA and protein levels, as determined by immunoblotting with highly specific anti-Id antibodies. Immunohistochemistry using these antibodies demonstrated the presence of faint Id-1 and Id-2 immunostaining in pancreatic ductal cells in the normal pancreas, whereas Id-3 immunoreactivity ranged from weak to strong. In the cancer tissues, many of the cancer cells exhibited abundant Id-1, Id-2, and Id-3 immunoreactivity. Scoring on the basis of percentage of positive cells and intensity of immunostaining indicated that Id-1 and Id-2 were increased significantly in the cancer cells by comparison with the respective controls. Mild to moderate Id immunoreactivity was also seen in the ductal cells in the CP-like areas adjacent to these cells and in the ductal cells of small and interlobular ducts in CP. In contrast, in dysplastic and atypical papillary ducts in CP, Id-1 and Id-2 immunoreactivity was as significantly elevated as in the cancer cells. These findings suggest that increased Id expression may be associated with enhanced proliferative potential of pancreatic cancer cells and of proliferating or dysplastic ductal cells in CP.

Bone morphogenetic protein 2 exerts diverse effects on cell growth in vitro and is expressed in human pancreatic cancer in vivo

BACKGROUND & AIMS Bone morphogenetic proteins (BMPs) belong to the transforming growth factor beta superfamily of signaling molecules. We characterized the expression of BMP-2 and its receptors in human pancreatic tissues and pancreatic cancer cell lines and examined the effects of BMP-2 on mitogenesis. METHODS Expression of BMP-2 and its receptors was determined by Northern blot analysis using specific complementary DNA probes. Distribution of BMP-2 in pancreatic cancers was examined by immunohistochemistry and in situ hybridization. Effects of BMP-2 on mitogenesis were assessed by monitoring cell proliferation and activation of mitogen-activated protein kinase (MAPK). RESULTS Compared with the normal pancreas, pancreatic cancers showed a 12.5-fold (P < 0.01), 2-fold (P < 0.01), and 8-fold (P < 0.01) increase of BMP-2, BMP receptor (R)-IA, and BMPR-II messenger RNA levels, respectively. By immunohistochemistry and in situ hybridization, BMP-2 was expressed in the cancer cells within the tumor mass. There was a significant correlation between the presence of BMP-2 immunostaining in the tumors and shorter postoperative survival. Pancreatic cancer cell lines expressed variable levels of messenger RNA encoding BMP-2 and its receptors. BMP-2 stimulated the growth of two pancreatic cancer cell lines (ASPC-1 and CAPAN-1). This mitogenic effect was associated with MAPK activation and blocked by the MAPK inhibitor PD98059 in CAPAN-1 but not in ASPC-1 cells. In both cell lines, expression of wild-type Smad4 abolished the BMP-2-mediated growth stimulation. BMP-2 inhibited the growth of COLO-357 cells, an effect that was blocked by expressing a dominant negative Smad4. BMP-2 had no effect in three cell lines that underexpressed either the BMP receptors or Smad1. CONCLUSIONS These findings indicate that BMP-2 has the capacity to act as a mitogen when Smad4 is mutated and suggest that it might play a role in the pathobiology of human pancreatic cancer.

Overexpression of activin A in stage IV colorectal cancer

BACKGROUND AND AIMS Activins and inhibins are dimeric polypeptides that belong to the transforming growth factor beta (TGF-β) superfamily and that bind to transmembrane receptors with serine/threonine kinase activity. The aim of this study was to characterise, in colon cancer cell lines and in normal and malignant human colon tissues, levels of expression of inhibin subunits that are involved in activin/inhibin dimer formation, and of the type I and II activin receptors (actRI and actRII). METHODS Expression of inhibin subunits and activin receptors was analysed by northern blot analysis. Inhibin βA and activin receptor expression were also assessed by use of polymerase chain reaction (PCR). In addition, activin A/inhibin βA localisation in human colon samples was assessed by immunohistochemistry and in situ hybridisation. RESULTS Inhibin βA mRNA was expressed in CaCo2 cells but not in SW 837 or SW 1463 cells whereas inhibin βB and inhibin α were below the level of detection. In contrast, all four activin receptors were present in the three cell lines. Colon cancers overexpressed inhibin βA mRNA in comparison with normal colon, and this overexpression was greatest in stage IV tumours. ActRIb mRNA levels were slightly higher in the normal colon than in cancer tissues. By immunohistochemistry and in situ hybridisation, activin A and inhibin βA mRNA were present in the mucosal epithelial cells in normal tissues from patients with stage I disease but were either absent or weakly present in normal tissues from patients with stage IV disease. Conversely, they were present at weak to moderate levels in stage I cancers but at high levels in stage IV cancers. CONCLUSIONS Our findings indicate that activin A is overexpressed in human colorectal tumours, especially in stage IV disease, raising the possibility that activin A may have a role in advanced colorectal cancer.

Coexpression of FAS and FAS-ligand in chronic pancreatitis: Correlation with apoptosis

Activation of the Fas receptor by Fas-ligand (FasL) results in apoptosis, and dysregulation of this pathway may contribute to abnormal cell proliferation and cell death. The aim of this study was to compare the expression of Fas and FasL in the normal pancreas and chronic pancreatitis (CP). By Northern blotting, Fas messenger RNA (mRNA) levels were increased in CP in comparison to the normal pancreas. Immunostaining revealed that faint Fas and FasL immunoreactivity was present in ductal and islet cells of the normal pancreas. In CP, there was faint Fas and strong FasL immunoreactivity in the proliferating ductal cells. Additionally, many of these ductal cells in the CP samples exhibited an apoptotic signal, as determined by DNA 3´-OH end labeling. These findings suggest that activation of apoptosis through the Fas receptor may contribute to the pathobiology of CP.