Kazuya Yamahatsu

Overexpressed fibroblast growth factor receptor 2 in the invasive front of colorectal cancer: A potential therapeutic target in colorectal cancer

Fibroblast growth factor receptor 2 (FGFR2) is considered a novel therapeutic target for various cancer. We used a silencing strategy to clarify the effect of reduced FGFR2 expression in human colorectal cancer (CRC) cells. The invasive front of cancer cells exhibited stronger FGFR2 expression than the surface area of the cancers. FGFR2 shRNA-transfected LoVo cells inhibited cell migration, invasion and tumor growth in vitro and in vivo. Thus, FGFR2 plays important roles in CRC progression in association with tumor cell migration, invasion and growth, and FGFR2 might be a novel therapeutic target for CRC.

Comparison of Fixation Methods for Preservation of Morphology, RNAs, and Proteins From Paraffin-Embedded Human Cancer Cell-Implanted Mouse Models

Xenograft transplantation of human tumor cells into immunodeficient mice is an important method to clarify the roles of specific molecules or chemicals in vivo. Recently, this method has been reported as a definitive examination to identify tumor stem cells. In this study, the authors compared the morphology and the quality and quantity of ribonucleic acid (RNA) and protein in paraffin-embedded tissues of nude mice implanted with human uterine cervical cancer cells, followed by fixation with commonly used fixatives, including 4% paraformaldehyde (PFA), 10% neutral buffered formalin (NBF), 20% NBF, and 99% ethanol (EtOH). The quality of the isolated RNA from PFA- and NBF-fixed paraffin-embedded tissues was high, while EtOH-fixed tissues showed degradation of RNA. NBF-fixed tissues showed excellent quality of morphology, but EtOH-fixed tissues showed contraction of cells. Immunohistochemical results showed differences depending on fixations. The 99% EtOH-fixed samples showed decreases of Ki-67 and VEGF-A immunoreactivities, but improved cytokeratin immunoreactivity. This study indicated that formalin fixation is better than alcohol fixation for RNA preservation in paraffin-embedded cancer cell implantation models. Immunohistochemical results differed markedly depending on fixation materials and antibodies; therefore, suitable fixations are needed to quantify and compare the results of immunohistochemical staining on cancer cell implanted nude mice tissues.

Epithelial splicing regulatory protein 1 is a favorable prognostic factor in pancreatic cancer that attenuates pancreatic metastases.

Epithelial splicing regulatory protein 1 (ESRP1) binds the FGFR-2 auxiliary cis-element ISE/ISS-3, located in the intron between exon IIIb and IIIc, and primarily promotes FGFR-2 IIIb expression. Here we assessed the role of ESRP1 in pancreatic ductal adenocarcinoma (PDAC). Immunohistochemical analysis was performed using anti-ESRP1, FGFR-2 IIIb and FGFR-2 IIIc antibodies in 123 PDAC cases. ESRP1 expression vector and small interference RNA (siRNA) targeting ESRP1 were transfected into human PDAC cells, and cell growth, migration and invasion were analyzed. In vivo heterotopic and orthotopic implantations using ESRP1 overexpression clones were performed and effects on pancreatic tumor volumes and hepatic and pulmonary metastases determined. ESRP1 immunoreactivity was strong in the nuclei of cancer cells in well-to-moderately differentiated PDACs but weak in poorly differentiated cancers. Well-to-moderately differentiated cancers also exhibited high FGFR-2 IIIb and low FGFR-2 IIIc expression, whereas this ratio was reversed in the poorly differentiated cancers. Increased ESRP1 expression was associated with longer survival in comparison with low ESRP1 expression, and PANC-1 cells engineered to express ESRP1 exhibited increased FGFR-2 IIIb expression and decreased migration and invasion in vitro, whereas ESRP1 siRNA-transfected KLM-1 cells exhibited increased FGFR-2 IIIc expression and increased cell growth, migration and invasion. In vivo, ESRP1-overexpressing clones formed significantly fewer liver metastases as compared with control clones. ESRP1 regulates the expression pattern of FGFR-2 isoforms, attenuates cell growth, migration, invasion and metastasis, and is a favorable prognostic factor in PDAC. Therefore, devising mechanisms to upregulate ESRP1 may exert a beneficial therapeutic effect in PDAC.

Nestin as a novel therapeutic target for pancreatic cancer via tumor angiogenesis

The class VI intermediate filament protein, nestin is reported to be a progenitor cell marker in various tissues. In the present study, we analyzed the expression and roles of nestin in angiogenesis of pancreatic ductal adenocarcinomas, and determined whether nestin is a potential target for inhibiting tumor angiogenesis using a gene silencing strategy. Nestin expression was detected only in small vessels, whereas CD34, CD31 and factor VIII were also expressed in large-sized blood vessels in PDAC. The number of nestin-positive vessels was approximately 20% the number of CD34-positive vessels, and the average dimension of nestin-positive vessels was approximately 75% that of CD34-positive vessels. The PCNA labeling indices of nestin-positive vessels were higher than those of CD34-positive vessels and nestin-negative vessels. Reducing nestin expression by use of siRNA targeting nestin transcripts inhibited growth of the vascular endothelial cell lines, but there was no difference in cell motility. In xenograft models, administration of siRNA targeting mouse-nestin suppressed subcutaneous human pancreatic cancer cell growth in nude mice. In conclusion, nestin was expressed in small proliferating blood vessels in pancreatic cancer tissues and may be a useful marker of angiogenesis in pancreatic ductal adenocarcinoma tissues. Furthermore, nestin is a potential novel therapeutic target in pancreatic cancers to inhibit tumor angiogenesis.

Pancreaticojejunostomy with closure of the pancreatic stump by endoscopic linear stapler in laparoscopic pancreaticoduodenectomy: a reliable technique and benefits for pancreatic resection.

We introduce a technique for pancreaticojejunostomy with closure of the pancreatic stump by endoscopic linear stapler as a reliable intervention with benefits for pancreatic resection in laparoscopic pancreaticoduodenectomy (Lap‐PD).

Clinical outcomes for 14 consecutive patients with solid pseudopapillary neoplasms who underwent laparoscopic distal pancreatectomy

The postoperative results of laparoscopic distal pancreatectomy for solid pseudopapillary neoplasm of the pancreas (SPN), including the effects of spleen‐preserving resection, are still to be elucidated.

Clinical outcomes of 15 consecutive patients who underwent laparoscopic insulinoma resection: The usefulness of monitoring intraoperative blood insulin during laparoscopic pancreatectomy

Insulinoma is a very serious functional tumor. Surgeons should confirm complete resection of insulinomas before completing the operation, even in laparoscopic surgery.

Nestin phosphorylation at threonines 315 and 1299 correlates with proliferation and metastasis of human pancreatic cancer

The neuroepithelial stem cell marker nestin is a cytoskeletal protein that regulates cell proliferation, invasion, and stemness in various tumors, including pancreatic tumors. In the present study, we examined the expression and roles of phosphorylated nestin in pancreatic cancer cells. Nestin phosphorylation at threonines 315 (Thr315) and 1299 (Thr1299) was observed during mitosis in human pancreatic cancer cells. Nestin phosphorylation was positively correlated with a cell proliferation marker, MIB‐1 expression in human pancreatic cancer samples. Transfection of MIA PaCa‐2 cells with nestin mutated at Thr315 and/or Thr1299 (to suppress phosphorylation) resulted in lower proliferation rates than those in control groups. Transfecting MIA PaCa‐2 cells with wild‐type nestin or with nestin mutated at Thr315 increased migration and invasion. In contrast, transfection with nestin mutated at both phosphorylation sites (Thr315 and Thr1299) did not enhance cell migration or invasion. In an intra‐splenic xenograft experiment using MIA PaCa‐2 cells, tumors expressing the nestin double mutant formed fewer liver metastases than tumors expressing wild‐type nestin. Nestin phosphorylation at these two sites was decreased upon treatment with inhibitors for cyclin dependent kinases, AKT, and Aurora in PANC‐1 cells, which express a high baseline level of phosphorylated nestin. These findings suggest that phosphorylation of nestin at Thr315 and/or Thr1299 affects cell proliferation, and inhibition of both phosphorylation sites suppresses invasion and metastasis of human pancreatic cancer. Inhibiting nestin phosphorylation at these two sites may represent a novel therapeutic strategy for pancreatic cancer.

Colonic stent-induced mechanical compression may suppress cancer cell proliferation in malignant large bowel obstruction

BackgroundThe short-term safety and efficacy of insertion of a self-expandable metallic colonic stent (SEMS) followed by elective surgery, “bridge to surgery (BTS)”, for malignant large bowel obstruction (MLBO) have been well described; however, the influence on long-term oncological outcomes is unclear. The aim of this study was to evaluate changes in oncological characteristics in colorectal cancer (CRC) tissues after SEMS insertion, focusing on growth factors, cell cycle and apoptosis.MethodsFrom January 2013 to September 2014, a total of 25 patients with MLBO who underwent BTS at our single institution were retrospectively included. Paired CRC tissue samples before (endoscopic biopsy) and after SEMS insertion (surgically resected) were collected from each patient. EGFR, VEGF, Ki-67, p27kip1 and TUNEL expression were determined by immunohistochemistry.ResultsNo clinical or subclinical perforations evaluated by mechanical ulceration pathologically were observed. Epithelial exfoliation, tumour necrosis, infiltration of inflammatory cells and fibrosis were observed in SEMS-inserted surgically-resected specimens. Overall, 84% (21/25) and 60% (15/25) of patients exhibited no change or a decrease in staining category, respectively, for EGFR and VEGF expression after SEMS insertion. A significant decrease in Ki-67 expression was observed in surgically-resected specimens compared with endoscopic biopsy specimens (P < 0.01). The upstream cell cycle inhibitor, p27kip1, was significantly increased after SEMS insertion (P = 0.049).ConclusionsAlthough the long-term safety of BTS should be determined in a future clinical trial, mechanical compression by SEMS may suppress cancer cell proliferation and this result could provide some insights into the issue.

A Case of Alpha-fetoprotein and Des-gamma-carboxy Prothrombin-Producing Gastric Carcinoma Mimicking Hepatocellular Carcinoma

A 77-year-old man presenting with diarrhea and upper abdominal pain was referred to our hospital owing to multiple liver nodules and bulky gastric lymph node noted on abdominal contrast computed tomography (CT). His serum alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) levels were 187,800 ng/mL and 135,000 mAU/mL, respectively. The liver tumors displayed an enhanced hepatocellular pattern on the contrast CT scan; however, there was no hepatitis viral infection or chronic liver cirrhosis. On contrast magnetic resonance imaging, the liver nodules showed high intensity on the T2-weighted image, and the differential diagnosis by CT was liver metastasis. Upper gastrointestinal endoscopy revealed an advanced gastric carcinoma (type 2) in the cardia, which was histopathologically diagnosed as gastric adenocarcinoma. The tumor cell expressed AFP, and histopathological findings of the liver tumor due to core needle biopsy were similar to those of the gastric lesion; however, the liver tumor was positive for AFP and DCP. Thus, the tumor was clinically diagnosed as a gastric carcinoma producing both AFP and DCP.