Masami Hattori

Hedgehog signaling pathway is a potential therapeutic target for gallbladder cancer

Gallbladder cancer (GBC) is a particularly deadly type of cancer with a 5‐year survival rate of only 10%. New effective therapeutic strategies are greatly needed. Recently, we have shown that Hedgehog (Hh) signaling is reactivated in various types of cancer and is a potential therapeutic target. However, little is known about the biological significance of Hh signaling in human GBC. In this study, we determined whether Hh signaling could be a therapeutic target in GBC. The Hh transcription factor Gli1 was detected in the nucleus of GBC cells but not in the nucleus of normal gallbladder cells. The expression levels of Sonic Hh (Shh) and Smoothened (Smo) in human GBC specimens (n = 37) were higher than those in normal gallbladder tissue. The addition of exogenous Shh ligand augmented the anchor‐dependent and anchor‐independent proliferation and invasiveness of GBC cells in vitro. In contrast, inhibiting the effector Smo decreased the anchor‐dependent and anchor‐independent proliferation. Furthermore, the suppression of Smo decreased GBC cell invasiveness through the inhibition of MMP‐2 and MMP‐9 expression and inhibited the epithelial–mesenchymal transition. In a xenograft model, tumor volume in Smo siRNA‐transfected GBC cells was significantly lower than in control tumors. These results suggest that Hh signaling is elevated in GBC and may be involved in the acquisition of malignant phenotypes, and that Hh signaling may be a potential therapeutic target for GBC.

Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial–mesenchymal transition

Anterior gradient 2 (AGR2), a member of the protein disulfide isomerase family, has been implicated in various cancers including pancreatic ductal adenocarcinoma (PDAC) and is known to promote cancer progression. However, the prognostic value of AGR2 expression and the interaction with epithelial–mesenchymal transition (EMT) remain unclear. We investigated the clinical significance of AGR2 and EMT markers in PDAC patients by immunohistochemical analyses. Although AGR2 expression was not observed in normal pancreas, all pancreatic precursor neoplastic lesions were positive for AGR2, even at the earliest stages, including pancreatic intraepithelial neoplasia-1A, AGR2 expression was reduced in 27.7% (54/195 cases) of PDAC patients. AGR2 downregulation correlated with EMT markers (vimentin overexpression and reduced membranous E-cadherin expression), high Union for International Cancer Control stage (P<0.0001), high histological cellular grade (P<0.0001), and adverse outcome (P<0.0001). In vitro, targeted silencing of AGR2 in cancer cells using siRNA reduced cell proliferation, colony formation, cell invasiveness, and migration, but did not alter EMT markers. To confer a more aggressive phenotype and induce EMT in PDAC cells, we co-cultured PDAC cell lines with primary-cultured pancreatic stellate cells (PSCs) and found that AGR2 was downregulated in co-cultured PDAC cells compared with PDAC monocultures. Treatment with transforming growth factor beta-1 (TGF-β), secreted from PSCs, decreased AGR2 expression, whereas inhibition of TGF-β signaling using recombinant soluble human TGF-β receptor type II and TGF-β-neutralizing antibodies restored AGR2 expression. We conclude that AGR2 downregulation is a useful prognostic marker, induced by EMT, and that secreted TGF-β from PSCs may partially contribute to AGR2 downregulation in PDAC patients. AGR2 downregulation does not induce EMT or a more aggressive phenotype, but is a secondary effect of these processes in advanced PDAC.

Differential ezrin and phosphorylated ezrin expression profiles between pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasm, and invasive ductal carcinoma of the pancreas

Intraductal papillary mucinous neoplasms (IPMNs) and pancreatic intraepithelial neoplasia (PanINs) are important premalignant lesions of pancreatic cancer. Ezrin is a member of the ezrin, radixin, and moesin protein family and acts as a cross-linker between the plasma membrane and the actin cytoskeleton. We investigated the roles of ezrin during carcinogenesis in IPMN and invasive ductal carcinoma and examined whether ezrin was a prognostic factor. We examined ezrin and phosphorylated ezrin (p-ezrin) expression in 131 IPMNs, 47 PanINs, and 59 invasive ductal carcinomas by immunohistochemical staining. Ezrin and p-ezrin (tyr354) expressions were significantly higher in IPMN with an associated invasive carcinoma, compared with those in IPMN with high-grade dysplasia (P = .03 and P = .0007, respectively). In all grades of PanINs, ezrin and p-ezrin (tyr353) were highly expressed. In patients with invasive ductal carcinoma, the presence of PanIN-2 or PanIN-3 was significantly correlated with positive ezrin and p-ezrin (tyr353) expression of the invasive ductal carcinoma component (P = .01 and P = .0004). The negative p-ezrin (tyr353) expression group of invasive ductal carcinoma showed a significantly worse prognosis than did the positive p-ezrin (tyr353) expression group by survival analysis (P = .04) and was a statistically significant adverse prognostic factor by both univariate and multivariate analyses (P = .048 and P = .015). Ezrin phosphorylation sites differ between the developments of IPMN and PanIN. Although p-ezrin (tyr354) expression in IPMNs is associated with tumor invasion, p-ezrin (tyr353) expression in invasive ductal carcinoma plays an important role not in tumor invasion and metastasis but in the early development of PanINs.

Pancreatic intraepithelial neoplasia in the background of invasive ductal carcinoma of the pancreas as a prognostic factor.

Of the recognized precursor lesions of pancreatic adenocarcinoma, pancreatic intraepithelial neoplasia (PanIN) is the most common form. However, little is known about the relationship between the grade of PanIN and prognosis for patients with invasive ductal carcinoma.

Peritoneal myofibroblasts at metastatic foci promote dissemination of pancreatic cancer

Myofibroblasts in the stroma of pancreatic cancers promote tumor proliferation, invasion and metastasis by increasing extracellular matrix and secretion of several growth factors. In contrast, the role of myofibroblasts at peritoneally disseminated sites of pancreatic cancer has not yet been determined. This study was designed to assess the role of myofibroblasts at peritoneally disseminated sites of pancreatic cancer. Three primary cultures of human peritoneal myofibroblasts (hPMFs) were established from disseminated sites of pancreatic cancer and their interactions with the SUIT-2 and CAPAN-1 human pancreatic cancer cell lines were analyzed in vitro. Using a model in BALB/c nu/nu mice, we compared the dissemination ability of intraperitoneally implanted pancreatic cancer cells, with and without hPMFs, and examined the presence of green fluorescent protein (GFP)-labeled hPMFs at peritoneally disseminated sites in mice. hPMFs significantly promoted the migration and invasion of pancreatic cancer cells (P<0.05), while the cancer cells significantly promoted the migration and invasion of hPMFs (P<0.05). In vivo, the number of peritoneally disseminated nodules, more than 3 mm in size, was significantly greater in mice implanted with cancer cells plus hPMFs compared to mice implanted with cancer cells alone, with GFP-labeled hPMFs surviving in the peritoneal cavity of the former. hPMFs promote the peritoneal dissemination of pancreatic cancer. The cancer-stromal cell interaction in the peritoneal cavity may be a new therapeutic target to prevent the dissemination of pancreatic cancer.

Follicular pancreatitis, report of a case clinically mimicking pancreatic cancer and literature review.

We herein present a 71-year-old man who underwent pancreatoduodenectomy with the diagnosis of follicular pancreatitis. We could not completely deny malignancy by a preoperative imaging study. Endoscopic ultrasonography-guided fine needle aspiration biopsy demonstrated clusters of benign acinar cells and no proliferation of atypical lymphoid cells or rich plasma cells. Histologically, the prominent lymphoid follicle formation was seen in an ill-defined mass, 15 mm in size, in the pancreatic parenchyma. Duct-centered fibrotic rims were seen in the pancreatic ducts accompanied by mild fibrotic change between the follicles and obliterative phlebitis. No neoplastic epithelial cells were observed in the resected specimen, and infiltrating lymphocytes did not show any morphological atypia and monoclonal proliferation by immunohistochemical staining with B and T cell markers. In addition, we could exclude IgG4-related disease, because plasmacytic cells were rarely positive for IgG4. Although follicular pancreatitis is rare, this mass-forming inflammatory disease (pancreatitis) should be included in the preoperative differential diagnosis of pancreatic cancer.

SLC2A1/GLUT1 expression in mural nodules of intraductal papillary mucinous neoplasm of the pancreas

In intraductal papillary mucinous neoplasms (IPMNs), the presence of a mural nodule showing a papillary or nodular proliferation of tumor cells in the dilated pancreatic duct is an indication for resection of IPMN. Solute carrier family 2, facilitated glucose transporter member 1, known as glucose transporter type 1 (SLC2A1/GLUT1) mediates cellular glucose uptake in many carcinomas and is correlated with increased 18F-fluorodeoxyglucose (18F-FDG) uptake. We examined SLC2A1/GLUT1 expression in the mural nodules of 180 IPMN specimens to distinguish malignant/benign tumors. A mural nodule was detected in 80 (44.4%) of the IPMNs, and was detected in 18.6% (13/70) of the IPMN-low (dysplasia) specimens, 36.1% (13/36) of the IPMN-int, 93.3% (28/30) of the IPMN-high, and 59.1% (26/44) of the IPMN-inv (with an associated invasive carcinoma) specimens. The sensitivity for detecting mural nodules was 81.7% by endoscopic ultrasonography, 70% by contrast-enhanced computed tomography and 54% by endoscopic retrograde cholangiopancreatography. SLC2A1/GLUT1 expression in the mural nodules was recognized in the basal and basolateral cytomembrane of tumor cells and was expressed in 15.4% (2/13) of the IPMN-low, 15.4% (2/13) of the IPMN-int, 71.4% (20/28) of the IPMN-high and 84.6% (22/26) of the IPMN-inv groups. The SLC2A1/GLUT1 expression was significantly higher in the IPMN-high and IPMN-inv mural nodules than in those of the IPMN-low and IPMN-int groups. Our findings suggest that SLC2A1/GLUT1 is expressed late in the adenoma-carcinoma sequence during carcinogenesis in IPMN, and SLC2A1/GLUT1 act as therapeutic target for malignant IPMN.