Satoshi Tanno

Natural History of Branch Duct Intraductal Papillary-Mucinous Neoplasms of the Pancreas without Mural Nodules: Long-term Follow-up Results

Background and aim: Although branch duct intraductal papillary-mucinous neoplasms (IPMNs) of the pancreas without mural nodules are frequently observed in asymptomatic subjects, the natural history of these lesions has never been studied. The aim of this study was to elucidate the natural history of branch duct IPMNs without mural nodules. Methods: Eighty-two patients who had no apparent mural nodules on initial examination were selected for follow-up. All subjects underwent examinations by imaging modalities including endoscopic retrograde pancreatography, and were followed-up by regular examinations once or twice a year. Serial changes of the maximum cystic diameter and the appearance of mural nodules were studied during the observation periods ranging from 14 to 148 months (median, 61 months). Results: Nine (11.0%) of 82 patients exhibited obvious progression of cystic dilatation (median, 59 months). Of these nine patients with cystic enlargement, six continued with regular follow-up examinations. Three cases underwent surgical resection, and were pathologically diagnosed as adenoma in two and borderline in one. Four patients (4.9%) showed newly developed mural nodules in dilated branch ducts (median, 105 months). Histological analysis revealed three cases classified as adenoma and one as carcinoma in situ. None of the remaining 69 patients (84.1%) showed any changes in dilated branch ducts (median, 57 months). Conclusions: Most branch duct IPMNs without mural nodules remained unchanged during long-term follow-up. Although follow-up with careful examination is required to detect newly developed mural nodules in dilated branch ducts, branch duct IPMNs without mural nodules can be followed-up without surgery.

Pancreatic ductal adenocarcinomas in long-term follow-up patients with branch duct intraductal papillary mucinous neoplasms.

Objective: Although branch duct intraductal papillary mucinous neoplasms (BD-IPMNs) are slow-growing tumors with a favorable prognosis, the synchronous occurrence of pancreatic ductal adenocarcinomas (PDAs) in patients with BD-IPMNs has been reported. This study was aimed to elucidate the development of PDAs in long-term follow-up patients with BD-IPMNs. Methods: We investigated 89 BD-IPMN patients who had no mural nodules and followed them up conservatively at least 2 years (median follow-up, 64 months; range, 25-158 months). All subjects underwent examinations by imaging modalities including endoscopic retrograde pancreatography. We calculated the standardized incidence ratio (SIR) from the vital statistics compiled by the Ministry of Health, Labor, and Welfare of Japan. Results: Among the 89 patients, 4 cases of PDAs distant from BD-IPMN were observed in 552 patient-years of follow-up (7.2 per 1000 patient-years). The expected number was 0.25, and the SIR of PDAs was 15.8 (95% confidence interval [CI], 4.3-40.4; P = 0.00014). Subgroup analyses showed that the incidence of PDAs was significantly increased in patients 70 years or older (SIR 16.7; 95% CI, 3.4-48.7; P = 0.0008) and in women (SIR 22.5; 95% CI, 2.7-81.1; P = 0.0037). Conclusions: Patients with BD-IPMNs are at a high risk for PDAs. During the follow-up, careful examination is required to detect the development of PDAs in patients with BD-IPMNs.Abbreviations: IPMN - intraductal papillary mucinous neoplasm, PDA - pancreatic ductal adenocarcinoma, ERCP - endoscopic retrograde cholangiopancreatography, MRCP - magnetic resonance cholangiopancreatography, EUS - endoscopic ultrasonography, CT - computed tomography, SIR - standardized incidence ratio

Clonality and field cancerization in intraductal papillary-mucinous tumors of the pancreas

Multiple lesions of intraductal papillary‐mucinous tumor of the pancreas (IPMT) in the same pancreas often are encountered. To elucidate field (multicentric) cancerization and clonality of IPMT, clonal analyses of IPMT and its precursor lesion of ductal hyperplasia were performed. K‐ras codon 12 mutations and X‐chromosome inactivation of human androgen receptor gene (HUMARA) were investigated.

Serine/Threonine Kinase AKT Is Frequently Activated in Human Bile Duct Cancer and Is Associated with Increased Radioresistance

The prognosis for patients with bile duct cancer (BDC) remains poor. Although BDC cells are essentially radioresistant, recent reports have suggested that radiation therapy, in addition to its palliative role in the management of BDC, may improve patient survival. A better understanding of the mechanisms that lead to cellular radioresistance may assist in the development of more effective BDC therapies based on radiotherapy in combination with radiosensitizing agents. The serine/threonine kinase AKT/protein kinase B, a downstream effector of phosphatidylinositol 3′-kinase, is a well-characterized kinase that is known to play a critical role in antiapoptotic signaling pathways. In this investigation, we sought to clarify the role of AKT signaling in the radioresistance in BDC cells. First, to examine whether activated AKT is expressed in BDCs, tumor specimens were obtained from 19 consecutive BDC cases. Immunohistochemical staining using an anti-phosphorylated-AKT antibody showed that phosphorylated (activated) AKT was expressed in cancer cells but not in neighboring normal mucosa in 16 cases (84.2%). Next, to evaluate the role of AKT activation in the regulation of BDC cell radiosensitivity, clonogenic assays were performed using the phosphatidylinositol 3′-kinase inhibitor LY294002 with and without irradiation. LY294002 inhibited AKT activation in BDC cells and, on irradiation, decreased clonogenic survival in a radiation dose-dependent manner. Only a small decrease in cell viability was observed in cells exposed to LY294002. Expression of constitutively active AKT in BDC cells resulted in decreased radiosensitivity, whereas a dominant-negative AKT increased radiosensitivity. Furthermore, constitutively active AKT also inhibited radiation-induced apoptosis. Collectively, these results indicate that activated AKT in BDC cells is associated with radioresistance and suggest that pharmacological or genetic modulation of AKT activity may have important therapeutic implications in BDC patients treated with radiation.

Hedgehog Promotes Neovascularization in Pancreatic Cancers by Regulating Ang-1 and IGF-1 Expression in Bone-Marrow Derived Pro-Angiogenic Cells

Background The hedgehog (Hh) pathway has been implicated in the pathogenesis of cancer including pancreatic ductal adenocarcinoma (PDAC). Recent studies have suggested that the oncogenic function of Hh in PDAC involves signaling in the stromal cells rather than cell autonomous effects on the tumor cells. However, the origin and nature of the stromal cell type(s) that are responsive to Hh signaling remained unknown. Since Hh signaling plays a crucial role during embryonic and postnatal vasculogenesis, we speculated that Hh ligand may act on tumor vasculature specifically focusing on bone marrow (BM)-derived cells. Methodology/Principal Findings Cyclopamine was utilized to inhibit the Hh pathway in human PDAC cell lines and their xenografts. BM transplants, co-culture systems of tumor cells and BM-derived pro-angiogenic cells (BMPCs) were employed to assess the role of tumor-derived Hh in regulating the BM compartment and the contribution of BM-derived cells to angiogenesis in PDAC. Cyclopamine administration attenuated Hh signaling in the stroma rather than in the cancer cells as reflected by decreased expression of full length Gli2 protein and Gli1 mRNA specifically in the compartment. Cyclopamine inhibited the growth of PDAC xenografts in association with regression of the tumor vasculature and reduced homing of BM-derived cells to the tumor. Host-derived Ang-1 and IGF-1 mRNA levels were downregulated by cyclopamine in the tumor xenografts. In vitro co-culture and matrigel plug assays demonstrated that PDAC cell-derived Shh induced Ang-1 and IGF-1 production in BMPCs, resulting in their enhanced migration and capillary morphogenesis activity. Conclusions/Significance We identified the BMPCs as alternative stromal targets of Hh-ligand in PDAC suggesting that the tumor vasculature is an attractive therapeutic target of Hh blockade. Our data is consistent with the emerging concept that BM-derived cells make important contributions to epithelial tumorigenesis.

PPARγ ligand-induced apoptosis through a p53-dependent mechanism in human gastric cancer cells

We have recently demonstrated that the PPARγ ligand troglitazone induced cell growth arrest and evoked apoptosis in a gastric cancer cell line, MKN–45. Since in general, p53 plays an important role in the induction of apoptosis and growth inhibition, we tried to clarify whether or not p53 mediates troglitazone‐induced apoptosis and growth arrest in gastric cancer cells. Troglitazone increased the number of apoptoic cells in MKN‐28, MKN‐45 and MKN‐74, but not in KATO‐III cells. The troglitazone‐induced apo‐ptotic change was significantly reduced by coincubation with bisphenol A digycidyl ether (BADGE), a synthetic PPARy antagonist, in MKN‐74 cells, suggesting that PPARγ mediates the apo‐ptotic effect of troglitazone. Since KATO‐III lacks the p53 gene, we speculated that p53 might be implicated in the PPARγ ligand‐induced apoptosis. Western blot analysis revealed that p53 expression was increased by troglitazone in a time‐dependent manner in MKN‐74 cells, further suggesting that p53 may mediate the ap‐optotic process induced by troglitazone. We next established a dominant‐negative p53 mutant by stable transfection of p53 mutant into MKN‐74 cells. In the dominant‐negative p53 mutant cells, troglitazone failed to induce apoptosis, strongly supporting the hypothesis that p53 indeed mediates the process of the troglitazone‐induced apoptosis. In the dominant‐negative p53 mutant cells, troglitazone significantly induced cell growth arrest and increased expression of p27Kip1 protein, which is thought to be the key molecule to evoke growth arrest, suggesting that p53 is not involved in the growth inhibition by troglitazone. All these results suggest that p53 mediates the PPARy ligand‐induced apoptosis, but not the cell growth inhibition. (Cancer Sci 2003; 94: 338–343)

Sonic hedgehog derived from human pancreatic cancer cells augments angiogenic function of endothelial progenitor cells

Hedgehog signaling is important in the pathogenesis of pancreatic cancer. Several recent observations suggest the involvement of sonic hedgehog (SHH) in postnatal neovascularization. We identified a novel role for SHH in tumor‐associated angiogenesis in pancreatic cancer. Immunohistochemical analysis revealed that patched homolog 1 (PTCH1), both a receptor for and transcriptional target of hedgehog signaling, was expressed in a small fraction of endothelial cells within pancreatic cancer, but not in normal pancreatic tissue. When endothelial progenitor cells (EPC) isolated from human peripheral blood were cultured with supernatant from SHH‐transfected 293 cells or pancreatic cancer cells, mRNA levels of vascular endothelial growth factor (VEGF), stromal cell‐derived factor‐1 and angiopoietin‐1 were significantly increased, whereas no such induction was observed in human umbilical vein endothelial cell (HUVEC) and human dermal microvascular endothelial cell (HMVEC). HUVEC tube formation was stimulated when cocultured with EPC, and preconditioning EPC with supernatant from KP‐1 N pancreatic cancer cells highly expressing SHH significantly enhanced the effect. The effect was partially attenuated by specific inhibition of SHH with cyclopamine or a neutralizing antibody. These findings suggest that tumor‐derived SHH can induce angiogenesis, and this is mediated by its effects on EPC specifically. Targeting SHH would be a novel therapeutic approach that can inhibit not only proliferation of cancer cells but also EPC‐mediated angiogenesis. (Cancer Sci 2008; 99: 1131–1138)

Growth arrest by troglitazone is mediated by p27Kip1 accumulation, which results from dual inhibition of proteasome activity and Skp2 expression in human hepatocellular carcinoma cells

In our study, we examined whether human hepatocellular carcinoma (HCC) expresses peroxisome proliferator‐activated receptor γ (PPARγ) and the effects of PPAR γ activation by its selective ligands on cell growth and cell invasion in HCC cells. RT‐PCR and Western blot analysis revealed that HCC‐derived cell lines, HepG2 and HLF, express PPARγ mRNA and protein. Luciferase assay in HLF cells showed that troglitazone, a selective ligand for PPAR γ, transactivated the transcription of a peroxisome proliferator response element‐driven promoter in a dose‐dependent manner, suggesting that the expressed PPARγ functions as a transcriptional factor. Not only troglitazone but pioglitazone dose‐dependently inhibited cell growth in HepG2 and HLF cells. Invasion assay using a transwell chamber demonstrated that troglitazone also inhibited cell invasion in HCC cells. To examine the mechanism of the troglitazone‐induced growth inhibition, we determined p27Kip1, a cyclin dependent kinase inhibitor, expression by Western blot analysis in troglitazone‐treated HLF cells. Troglitazone increased p27Kip1 in time‐ and dose‐dependent manners, suggesting that p27Kip1 may be involved in the growth inhibition by troglitazone in HLF cells. To further examine the mechanism of the troglitazone‐induced p27Kip1 protein accumulation, 2 major systems for regulation of p27Kip1 protein, proteasome activity and Skp2, an F‐box protein that targets p27Kip1 for degradation, were evaluated. Troglitazone potently inhibited proteasome activity and decreased Skp2 protein levels. All these results suggest that human HCC cells express functional PPAR γ and PPARγ activation resulted in growth inhibition. The growth inhibition was mediated by p27Kip1 accumulation, which is induced by both inhibition of ubiquitylation of p27Kip1 and reduction of degradation activity of p27Kip1 by proteasome.

Inhibition of cell invasion and morphological change by troglitazone in human pancreatic cancer cells.

BackgroundWe have recently demonstrated that peroxisome proliferator activated receptor (PPAR) γ activation by its selective ligand, troglitazone, potently inhibited cell proliferation in human pancreatic cancer cells. The present study was performed to clarify the role of PPARγ in cell invasion/motility in human pancreatic cancer cells.MethodsCell invasive activity was assessed by an in vitro invasion assay, using a Transwell chamber, and by a wound-healing assay, in the human pancreatic cancer cell lines, PK-1 and PK-9. Cell morphology and actin structure were evaluated by phase-contrast and fluorescence microscopy.ResultsPPARγ activation by troglitazone inhibited cell invasion and cell migration in PK-1 and PK-9 cells. We also examined the effect of troglitazone on cell morphology and actin structure because of its effect on cell motility. The size of PK-1 and PK-9 cells that had been incubated with troglitazone became smaller, and the in shape changed from flat to spindle, followed by round. The troglitazone-induced cell rounding was reversible by replacement with troglitazone-free medium. Rhodamine-phalloidin staining revealed a decreased number of actin filaments in PK-1 cells treated with troglitazone. In cells treated with mycalolide B, an actin depolymerizing agent, troglitazone failed to induce cell rounding.ConclusionsThese results suggest that PPARγ activation by troglitazone inhibited cell motility and changed cell morphology through modulating actin organization.

Solitary true cyst of the pancreas in two adults: analysis of cyst fluid and review of the literature

Solitary true cyst of the pancreas in adults is extremely rare. We report two adult cases of solitary true cyst of the pancreas. In a 53-yr-old woman there was discovered, incidentally, a unilocular cyst of 7.0 × 6.5 cm in size in the tail of the pancreas that was noted on abdominal US and CT scan. A 16-yr-old boy presented with abdominal pain, and an abdominal US and CT scan revealed a 6.5 cm cystic mass located in the tail of the pancreas. Both patients underwent distal pancreatectomy. Histologically, the cyst was lined with flattened cuboidal and squamous epithelium without morphological alterations. Analysis of the cyst fluid revealed high CA 19-9 (>100,000 U/L) and Span-1 levels (>60,000 U/L) in both cases. Immunohistochemically, the lining epithelial cells of true cyst were positive for CA 19-9 staining. The clinicopathological features of solitary true cyst of the pancreas in adults are briefly reviewed.