Toshiyuki Ishiwata

The cell-surface heparan sulfate proteoglycan glypican-1 regulates growth factor action in pancreatic carcinoma cells and is overexpressed in human pancreatic cancer.

Heparan sulfate proteoglycans (HSPGs) play diverse roles in cell recognition, growth, and adhesion. In vitro studies suggest that cell-surface HSPGs act as coreceptors for heparin-binding mitogenic growth factors. Here we show that the glycosylphosphatidylinositol- (GPI-) anchored HSPG glypican-1 is strongly expressed in human pancreatic cancer, both by the cancer cells and the adjacent fibroblasts, whereas expression of glypican-1 is low in the normal pancreas and in chronic pancreatitis. Treatment of two pancreatic cancer cell lines, which express glypican-1, with the enzyme phosphoinositide-specific phospholipase-C (PI-PLC) abrogated their mitogenic responses to two heparin-binding growth factors that are commonly overexpressed in pancreatic cancer: fibroblast growth factor 2 (FGF2) and heparin-binding EGF-like growth factor (HB-EGF). PI-PLC did not alter the response to the non-heparin-binding growth factors EGF and IGF-1. Stable expression of a form of glypican-1 engineered to possess a transmembrane domain instead of a GPI anchor conferred resistance to the inhibitory effects of PI-PLC on growth factor responsiveness. Furthermore, transfection of a glypican-1 antisense construct attenuated glypican-1 protein levels and the mitogenic response to FGF2 and HB-EGF. We propose that glypican-1 plays an essential role in the responses of pancreatic cancer cells to certain mitogenic stimuli, that it is relatively unique in relation to other HSPGs, and that its expression by pancreatic cancer cells may be of importance in the pathobiology of this disorder.

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.

Concomitant over-expression of vascular endothelial growth factor and its receptors in pancreatic cancer.

Vascular endothelial growth factor (VEGF) is a potent angiogenic polypeptide that activates 2 distinct high‐affinity tyrosine kinase receptors, flk‐1/KDR and flt‐1. In the present study, we characterized the expression of VEGF and its receptors flk‐1/KDR and flt‐1 in the normal human pancreas and in human pancreatic cancer tissues and cell lines. VEGF, flk‐1/KDR and flt‐1 mRNA levels were elevated in cancer tissues compared with normal pancreas. By immuno‐histochemistry, VEGF, flk‐1/KDR and flt‐1 immunoreactivity co‐localized in many of the cancer cells within the tumor mass. Three (AsPC‐1, Capan‐1 and MIAPaCa‐2) of 6 pancreatic cancer cell lines expressed flk‐1/KDR mRNA and protein, and 4 cell lines (AsPC‐1, Capan‐1, T3M4 and PANC‐1) expressed flt‐1 mRNA transcripts. Binding studies with 125I‐labeled VEGF165 indicated that only Capan‐1 cells exhibited high levels of specific binding. Furthermore, VEGF enhanced the growth of Capan‐1 cells but was without effect in the other cell lines. VEGF also enhanced mitogen‐activated protein kinase (MAPK) phosphorylation and c‐fos induction in Capan‐1 cells, whereas the MAPK kinase inhibitor PD98059 abolished the growth‐stimulatory effect of VEGF. These data indicate that human pancreatic cancers have the capacity to over‐express VEGF and its receptors and suggest that in some instances VEGF may directly promote pancreatic cancer growth via the MAPK pathway. Int. J. Cancer 85:27–34, 2000.

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.

INSULIN-LIKE GROWTH FACTOR I IMPROVES CARDIOVASCULAR FUNCTION AND SUPPRESSES APOPTOSIS OF CARDIOMYOCYTES IN DILATED CARDIOMYOPATHY

To investigate how insulin-like growth factor I (IGF-I) modulates cardiovascular function and myocardial apoptosis in heart failure, the therapeutic effects of IGF-I were determined in a canine model of dilated cardiomyopathy. The animals were paced at 220 beats/min, and the left ventricular (LV) chamber became dilated after 2 weeks. A subset of paced dogs was treated with sc injections of IGF-I from week 3 to week 4. After 4 weeks of pacing, untreated paced dogs developed significant ventricular dysfunction. IGF-I-treated paced dogs showed better cardiac output, stroke volume, LV end-systolic pressure, and LV end-diastolic pressure. Moreover, pulmonary wedge pressure and systemic vascular resistance were increased in the untreated group and decreased in the IGF-I-treated group. IGF-I treatment was associated with less thinning of the ventricular wall. Compared with the controls, untreated paced dogs showed increased apoptosis of cardiac muscle cells, which was partially suppressed by IGF-I treatment. The...

Fibroblast growth factor-5 stimulates mitogenic signaling and is overexpressed in human pancreatic cancer: evidence for autocrine and paracrine actions.

Fibroblast growth factor (FGF)-1 and -2 are overexpressed in human pancreatic cancer. In this study the role of FGF-5 in human pancreatic cancer was investigated, as FGF-5 has a classical signal sequence for secretion not found in FGF-1 or -2. Northern blot analysis with a 306 bp FGF-5 cDNA revealed the presence of 4.0 kb and 1.6 kb FGF-5 mRNA transcripts in both normal and cancerous pancreatic tissues. Densitometric analysis indicated that 4.0 kb and 1.6 kb FGF-5 mRNA transcripts levels were increased 2.4- and 2.7-fold in the cancers by comparison with normal tissues, respectively (P<0.002, P<0.0001). Immunohistochemistry and in situ hybridization demonstrated that FGF-5 localized in the cancer cells, stromal fibroblast and inflitrating macrophages. FGF-5 mRNA was also detected in COLO-357 human pancreatic cancer cells. Furthermore, secreted FGF-5 protein was present in conditioned medium of COLO-357 cells. Exogeneous FGF-5 (0.37 nM) increased the growth of COLO-357 cells by 48% (P<0.0001) and increased mitogen-activated protein kinase activity. COLO-357 cells expressed the IIIc isoform of the type I FGF receptor, the preferred FGF receptor for FGF-5. These observations suggest that FGF-5 may participate in autocrine and paracrine pathways promoting pancreatic cancer cell growth in vivo.

Increased Cyclin D1 in Human Pancreatic Cancer Is Associated with Decreased Postoperative Survival

Cyclin D1 belongs to a family of protein kinases that have been implicated in cell cycle regulation. In the present study we characterized cyclin D1 expression in 6 cultured human pancreatic cancer cell lines and in normal and cancerous human pancreatic tissues. A 4.4-kb cyclin D1 mRNA transcript was present in all cell lines and in all pancreatic tissues. Cyclin D1 mRNA levels were 2.1-fold higher in the pancreatic cancers than in normal pancreatic tissues (p < 0.0002). Cancer patients with lower cyclin D1 levels (n = 16) had a median survival of 15.5 months whereas patients with higher levels (n = 16) had a median survival of 6.5 months (p < 0.007). These data indicate that cyclin D1 expression may serve as a predictor of postoperative survival in pancreatic cancer patients, and raise the possibility that treatment modalities blocking cyclin D1 activity may have a future role in the therapy of these patients.

Concomitant over‐expression of activin/inhibin β subunits and their receptors in human pancreatic cancer

Activins and inhibins belong to the transforming growth factor‐β (TGF‐β) superfamily of multifunctional cytokines that bind to transmembrane receptors with serine/threonine kinase activity. In this study, we characterized the levels of expression of 3 activin/inhibin subunits (βA, βB, α), and 2 type I and type II activin receptors (actRI/Ib, actRII/IIb) in pancreatic cancer cell lines and in human pancreatic tissues. In addition, we assessed the growth responsiveness to activin A in these cell lines. All 6 cell lines (ASPC‐1, CAPAN‐1, COLO‐357, MIA‐PaCa‐2, PANC‐1 and T3M4) expressed the activin/inhibin βA subunit, whereas expression levels of the activin/inhibin βB and α subunits were undetectable. Furthermore, actRI, actRII and actRIIb were expressed in all cell lines and actRIb mRNA was evident in ASPC‐1, CAPAN‐1, COLO‐357 and PANC‐1 cells. CAPAN‐1 and COLO‐357 cells were growth‐stimulated by activin A in the presence of 10% serum, whereas the other cell lines were resistant to activin A. In contrast, in serum‐free medium activin A inhibited the growth of CAPAN‐1, COLO‐357 and MIA‐PaCa‐2 cells. Pancreatic cancer samples markedly over‐expressed the activin/inhibin βA subunit, whereas the βB subunit was only moderately increased in comparison to normal pancreatic samples. Pancreatic cancer tissues also markedly over‐expressed actRI, actRIb and actRII. By in situ hybridization, activin/inhibin βA, actRI, actRIb and actRII were strongly expressed in diffuse infiltrative and duct‐like cancer cells. Both the ligand and its receptors were often co‐expressed in these cells. Together, our findings suggest that activin A may participate in autocrine activation of pancreatic cancer cells in vivo. Int. J. Cancer 77:860–868, 1998.© 1998 Wiley‐Liss, Inc.

Amphiregulin antisense oligonucleotide inhibits the growth of T3M4 human pancreatic cancer cells and sensitizes the cells to EGF receptor-targeted therapy

Human pancreatic cancers overexpress the epidermal growth factor (EGF) receptor (EGFR) and all 5 ligands that bind to this receptor, including amphiregulin. It is not known, however, whether amphiregulin contributes in an autocrine manner to enhance pancreatic cancer cell growth. Therefore, we used an amphiregulin antisense oligonucleotide (AR‐AS) to suppress amphiregulin expression in T3M4 human pancreatic cancer cells. These cells express high levels of EGFR and amphiregulin. AR‐AS abolished amphiregulin immunoreactivity in T3M4 cells, decreased amphiregulin release into the medium and inhibited cell growth in a dose‐dependent manner. Exogenous amphiregulin reversed AR‐AS‐mediated growth inhibition. A random oligonucleotide (AR‐R) did not alter either cell growth or cellular amphiregulin immunoreactivity. AR‐AS also increased cellular EGFR protein levels and enhanced the growth‐inhibitory actions of TP40, a chimeric protein consisting of transforming growth factor‐α coupled to Pseudomonas exotoxin that internalizes into cells via EGFR. These findings indicate that there is an important EGFR/amphiregulin autocrine loop in T3M4 cells and raise the possibility that modalities aimed at abrogating amphiregulin action may prove useful in pancreatic cancer, especially when used in conjunction with EGFR‐targeted therapy. Int. J. Cancer 72:512–517, 1997.

Differential localization of transforming growth factor-β isoforms in human gastric mucosa and overexpression in gastric carcinoma

Transforming growth factor β (TGF‐β) isoforms comprise a family of multifunctional polypeptide growth factors that either inhibit or stimulate cell proliferation. We examined TGF‐β expression in normal human gastric mucosa and carcinoma. The distribution and expression of TGF‐β isoforms in 4 normal mucosa samples from organ donors, in 12 normal mucosa samples adjacent to gastric cancer and in 12 gastric carcinomas were examined using immunohistochemistry and Northern blot analysis. Because TGF‐βs regulate collagen expression, collagen type 1 α1 mRNA amounts were also examined. Immunohistochemical analysis of normal human gastric tissue samples indicated that TGF‐β1 localized principally in parietal cells but also in some surface mucus cells, TGF‐β2 was present exclusively in chief cells and TGF‐β3 was present in parietal, chief and mucus cells. In the gastric cancers, strong colocalization of TGF‐β1, ‐β2 and ‐β3 was evident in the cancer cells. Northern blot analysis indicated that, compared to normal gastric tissue, gastric cancers showed a 4.8‐ and 6‐fold increase in mRNA amounts encoding TGF‐β1, and TGF‐β3, respectively. In contrast, TGF‐β2 mRNA amounts were comparable in both groups. Northern blot analysis showed a 10‐fold increase in human collagen type 1 α1 mRNA amounts compared to normal gastric tissue. These findings imply a role for TGF‐βs in normal human gastric mucosa function, and raise the possibility that the aberrant colocalization and overexpression of all 3 TGF‐β isoforms in human gastric cancer cells in vivo may bcontribute to the pathobiology of gastric carcinoma. Int. J. Cancer 71:131–137, 1997.