Kenoki Ohuchida

Radiation to stromal fibroblasts increases invasiveness of pancreatic cancer cells through tumor-stromal interactions.

Radiotherapy represents a major treatment option for patients with pancreatic cancer, but recent evidence suggests that radiation can promote invasion and metastasis of cancer cells. Interactions between cancer cells and surrounding stromal cells may play an important role in aggressive tumor progression. In the present study, we investigated the invasive phenotype of pancreatic cancer cells in response to coculture with irradiated fibroblasts. Using in vitro invasion assay, we demonstrated that coculture with nonirradiated fibroblasts significantly increased the invasive ability of pancreatic cancer cells and, surprisingly, the increased invasiveness was further accelerated when they were cocultured with irradiated fibroblasts. The hepatocyte growth factor (HGF) secretion from fibroblasts remained unchanged after irradiation, whereas exposure of pancreatic cancer cells to supernatant from irradiated fibroblasts resulted in increased phosphorylation of c-Met (HGF receptor) and mitogen-activated protein kinase activity, possibly or partially via increased expression of c-Met. We also demonstrated that scattering of pancreatic cancer cells was accelerated by the supernatant from irradiated fibroblasts. The enhanced invasiveness of pancreatic cancer cells induced by coculture with irradiated fibroblasts was completely blocked by NK4, a specific antagonist of HGF. These data suggest that invasive potential of certain pancreatic cancer cells is enhanced by soluble mediator(s) released from irradiated fibroblasts possibly through up-regulation of c-Met expression/phosphorylation and mitogen-activated protein kinase activity in pancreatic cancer cells. Our present findings further support the potential use of NK4 during radiotherapy for patients with pancreatic cancer.

MicroRNA-21 modulates biological functions of pancreatic cancer cells including their proliferation, invasion, and chemoresistance.

Due to the poor prognosis of pancreatic cancer, novel diagnostic modalities for early diagnosis and new therapeutic strategy are urgently needed. Recently, microRNA-21 (miR-21) was reported to be strongly overexpressed in pancreatic cancer as well as in other solid cancers. We investigated the functional roles of miR-21, which have not been fully elucidated in pancreatic cancer. miR-21 expression was assessed in pancreatic cancer cell lines (14 cancer cell lines, primary cultures of normal pancreatic epithelial cells and fibroblasts, and a human normal pancreatic ductal epithelial cell line) and pancreatic tissue samples (25 cancer tissues and 25 normal tissues) by quantitative real-time reverse transcription-PCR amplification. Moreover, we investigated the proliferation, invasion, and chemoresistance of pancreatic cancer cells transfected with miR-21 precursor or inhibitor. miR-21 was markedly overexpressed in pancreatic cancer cells compared with nonmalignant cells, and miR-21 in cancer tissues was much higher than in nonmalignant tissues. The cancer cells transfected with the miR-21 precursor showed significantly increased proliferation, Matrigel invasion, and chemoresistance for gemcitabine compared with the control cells. In contrast, inhibition of miR-21 decreased proliferation, Matrigel invasion, and chemoresistance for gemcitabine. Moreover, miR-21 positively correlated with the mRNA expression of invasion-related genes, matrix metalloproteinase-2 and -9, and vascular endothelial growth factor. These data suggest that miR-21 expression is increased in pancreatic cancer cells and that miR-21 contributes to the cell proliferation, invasion, and chemoresistance of pancreatic cancer.[Mol Cancer Ther 2009;8(5):1067–74]

MicroRNA, hsa-miR-200c, is an independent prognostic factor in pancreatic cancer and its upregulation inhibits pancreatic cancer invasion but increases cell proliferation

BackgroundRecently, the microRNA-200 family was reported to affect cancer biology by regulating epithelial to mesenchymal transition (EMT). Especially, the expression of miR-200c has been shown to be associated with upregulating the expression of E-cadherin, a gene known to be involved in pancreatic cancer behavior. However, the significance of miR-200c in pancreatic cancer is unknown.MethodsIn the present study, we investigated the relationship between E-cadherin and miR-200c expression in a panel of 14 pancreatic cancer cell lines and in macro-dissected formalin-fixed paraffin-embedded (FFPE) tissue samples obtained from 99 patients who underwent pancreatectomy for pancreatic cancer. We also investigated the effects of miR-200c on the proliferation and invasion of pancreatic cancer cells.ResultsWe found that patients with high levels of miR-200c expression had significantly better survival rates than those with low levels of miR-200c expression. We also found a remarkably strong correlation between the levels of miR-200c and E-cadherin expression.ConclusionsThese data indicate that miR-200c may play a role in the pancreatic cancer biology and may be a novel marker for the prognosis of pancreatic cancer.

The Role of S100A6 in Pancreatic Cancer Development and Its Clinical Implication as a Diagnostic Marker and Therapeutic Target

Recent microarray analyses showed that the S100 family contains members that are candidate diagnostic markers or therapeutic targets. In the present study, to evaluate the involvement of S100A6 in pancreatic cancer and its clinical usefulness for diagnosis, we examined S100A6 mRNA expression in pancreatic tissues and pancreatic juice from patients with different pancreatic diseases. To investigate the role of S100A6 in carcinogenesis of pancreatic cancer and the potential of S100A6 as a diagnostic marker for early detection of pancreatic cancer, we did immunohistochemistry and microdissection-based mRNA analysis of pancreatic normal ducts, pancreatic intraepithelial neoplasias, and invasive ductal carcinomas. We also used in vitro experiments and microarray analysis with RNA interference to evaluate the functional role of S100A6 and its potential as a therapeutic target for pancreatic cancer. S100A6 mRNA levels were significantly higher in carcinoma specimens than in nonneoplastic tissues. In pancreatic juice, there was a significant difference in S100A6 expression between patients with carcinoma and those with nonneoplastic disease. Receiver operating characteristic curves revealed that S100A6 might be a useful marker for diagnosis of pancreatic cancer. Immunohistochemistry and microdissection-based analysis showed differential expression of S100A6 among normal ducts, pancreatic intraepithelial neoplasias, and invasive ductal carcinomas. In vitro data showed that inhibition of S100A6 decreased proliferation and invasiveness of cancer cells, and these findings were supported by microarray data. Our present results suggest that quantitation of S100A6 mRNA is a promising tool for diagnosis of pancreatic cancer, and that S100A6 may be a promising therapeutic target for pancreatic cancer.

Laparoscopy-Assisted Distal Gastrectomy for Early Gastric Cancer: Is It Beneficial for Patients of Heavier Weight?

Objective: In this retrospective review, we evaluated the advantages and disadvantages of LADG for patients of heavier weight with early gastric cancer. Summary Background Data: LADG has been used to treat early gastric cancer. We and others have reported less operative blood loss, less pain, early recovery of bowel activity, early restart of oral intake, and a shorter hospital stay with LADG compared with a conventional open method. There is, however, little information on the advantages of LADG for obese patients with early gastric cancer. Methods: Between January 1996 and March 2002, 76 patients with preoperatively diagnosed early gastric carcinoma underwent LADG in our department. We classified these patients into 2 groups on the basis of body mass index (BMI). Nineteen patients had a high-BMI (≥ 24.2 kg/m2), and 57 patients had a normal-BMI (<24.2 kg/m2). We collected data by retrospectively reviewing the medical charts. Results: Extension of the minilaparotomic incision or conversion to laparotomy was needed in 6 (32%) of the 19 patients in the high-BMI group, whereas only 3 (5%) of 57 patients in the normal-BMI group required either. In the high-BMI group, Roux-en-Y anastomosis rather than Billroth I anastomosis was adopted more often than in the normal-BMI group, due to the difficulty of the reconstruction (58% versus 4%, P = 0.001). Significantly longer operative time (370 ± 61 minutes versus 317 ± 58 minutes, P = 0.015) and prolonged recovery of bowel activity (3.5 ± 1.0 days versus 2.6 ± 1.0 days, P = 0.007) were observed in the patients in the high-BMI group. Conclusions: In the current study, LADG in patients of heavier weight was accompanied by more technical difficulties, and the disadvantages of longer operative time and delayed recovery of bowel activity was observed in patients of heavier weight. Heavier weight appears to be an ominous factor in the successful completion of LADG and should be considered in the decision to use LADG. There are still benefits of a decreased incidence of serious wound and hernia complications in successful cases.

MicroRNA-10b is overexpressed in pancreatic cancer, promotes its invasiveness, and correlates with a poor prognosis

BACKGROUND MicroRNAs (miRNAs) have been gaining attention as new, key molecules that contribute to carcinogenesis. In pancreatic cancer, previous profiling analyses of miRNA expression have shown that several miRNAs are differently expressed in normal and cancerous tissues. Several pancreatic cancer-specific miRNAs differed, however, in each analysis. METHODS We investigated the miRNA expression profiles of the pancreatic cancer cell lines CAPAN-1 and CFPAC1 and an immortalized human normal pancreatic ductal epithelial cell line (HPDE) using a high-throughput, TaqMan, qRT-PCR array analysis. We also analyzed the expression levels of this miRNA in microdissected (n = 15) and formalin-fixed, paraffin-embedded (FFPE) (n = 115) samples from pancreatic cancers by quantitative RT-PCR. Finally, we investigated the effects of this miRNA on the invasiveness of pancreatic cancer cells. RESULTS Based on the microarray analysis, miR-372, miR-146a, miR-204, miR-10a, and miR-10b showed particularly large differences (>10-fold changes) between both pancreatic cell lines and HPDE cells. Thirteen of the 15 pancreatic cancer cell lines showed 2.1- to 36.4-fold (median, 15.3-fold) greater levels of miR-10b than HPDE cells. Microdissection analysis revealed that miR-10b exhibited greater expression levels in pancreatic cancer cells (n = 5) than in normal pancreatic ductal cells (n = 10) (P < .020). Analysis of FFPE samples showed that high miR-10b expression was associated with a lesser overall survival (P = .014). Furthermore, miR-10b correlated with the invasiveness of pancreatic cancer cells (P < .01). CONCLUSION miR-10b is overexpressed in pancreatic cancer and may be involved in the invasiveness in pancreatic cancer cells, thereby leading to a poor prognosis.

CD10+ pancreatic stellate cells enhance the progression of pancreatic cancer

BACKGROUND & AIMS Pancreatic stellate cells (PSCs) promote the progression of pancreatic cancer by producing extracellular matrix and soluble factors. However, the functional heterogeneity of PSCs has not been identified until now. Detailed characterization of the PSCs in human pancreatic cancer would provide a set of potential targets for stroma-directed therapy. METHODS We isolated PSCs from fresh pancreatic ductal adenocarcinoma tissue and sorted them by flow cytometry according to cell surface expression of CD10, which is a stromal prognostic marker for various tumors. We analyzed the functional differences between CD10(+) PSCs and CD10(-) PSCs. RESULTS Immunohistochemical analysis showed that the frequency of CD10 expression by PSCs was markedly higher in tumor tissue than in normal tissue (33.7% vs 0%, respectively, P = .028). In pancreatic ductal adenocarcinoma, CD10 expression by PSCs was associated with positive nodal metastases (P = .011) and a shorter survival time (P < .001). In vitro coculture experiments showed that CD10(+) PSCs promoted the invasiveness of pancreatic cancer cell lines, SUIT-2 and Panc-1 cells more intensively than CD10(-) PSCs. CD10(+) PSCs significantly increased the tumor growth and invasiveness of SUIT-2 cells in a murine cotransplantation model. CD10(+) PSCs secreted higher levels of matrix metalloproteinase 3 than CD10(-) PSCs, and knockdown of matrix metalloproteinase 3 in cocultured PSCs reduced the invasion of SUIT-2 and Panc-1 cells. CONCLUSIONS CD10(+) PSCs enhance the progression of pancreatic cancer cells. CD10(+) PSCs may be a candidate for selective therapeutic targeting in the treatment of pancreatic cancer.

S100P Is an Early Developmental Marker of Pancreatic Carcinogenesis

Purpose: Our goal was to clarify the involvement and clinical significance of S100P in pancreatic carcinogenesis. Experimental Design: We examined S100P expression in 45 bulk pancreatic tissues; in microdissected cells, including invasive ductal carcinoma (IDC) cells (20 sections), pancreatic intraepithelial neoplasia (PanIN) cells (12 sections), intraductal papillary mucinous neoplasm (IPMN) cells (19 sections), and normal epithelial cells (11 sections); and in pancreatic juice samples from 99 patients with pancreatic diseases (32 cancer, 35 IPMN, and 32 chronic pancreatitis samples). We used quantitative real-time reverse transcription-PCR with gene-specific priming to measure S100P in these various types of samples. Results: In bulk tissue analyses, pancreatic cancer and IPMN expressed significantly higher levels of S100P than did nonneoplastic pancreas (P < 0.017 and P = 0.0013, respectively). Microdissection analyses revealed that IPMN expressed significantly higher levels of S100P than did IDC (P < 0.0001) and PanIN (P = 0.0031), although S100P expression did not differ between IDC and PanIN (P = 0.077). In pancreatic juice analyses, cancer and IPMN juice expressed significantly higher levels of S100P than did pancreatitis juice (both P < 0.0001). Receiver operating characteristic curve analyses revealed that measurement of S100P in pancreatic juice was useful for discriminating neoplastic disease from chronic pancreatitis (area under the curve = 0.837; 95% confidence interval, 0.749-0.903). Conclusion:S100P may be an early developmental marker of pancreatic carcinogenesis, and measurement of S100P in pancreatic juice may be useful for early detection of pancreatic cancer or screening of early pancreatic carcinogenesis.

Invasive carcinoma derived from the nonintestinal type intraductal papillary mucinous neoplasm of the pancreas has a poorer prognosis than that derived from the intestinal type.

BACKGROUND Intraductal papillary mucinous neoplasm (IPMN) of the pancreas is divided into 4 subtypes: an intestinal type, a gastric type, a pancreatobiliary type, and an oncocytic type. The purposes of this study were to clarify the outcomes and the characteristics of invasive carcinoma derived from IPMN (invasive IPMC) by focusing on these subtypes with a comparison to conventional invasive ductal carcinoma (IDC) of the pancreas. METHODS A total of 30 patients with invasive IPMC were reviewed, and the tumors were divided into 2 pathologic subtypes, intestinal and nonintestinal type. The prognosis and characteristics of the 2 subtypes were evaluated. Furthermore, the prognosis of 119 patients with conventional IDC was compared with that of patients with invasive carcinoma derived from the intestinal or nonintestinal type IPMN. RESULTS The 5-year survival rate of patients with the nonintestinal type (0.0%) was as poor as that of patients with conventional IDC (19.9%; P = .67). The patients with the intestinal type (66.7%) had a more favorable prognosis than patients with conventional IDC (P < .001). The nonintestinal type was characterized by positive lymphatic invasion and tubular invasive pattern. CONCLUSION Invasive carcinoma derived from the nonintestinal type IPMN characterized by lymphatic invasion and tubular invasive pattern is associated with a poor prognosis.

Intraductal Papillary Mucinous Neoplasms of the Pancreas With Distinct Pancreatic Ductal Adenocarcinomas Are Frequently of Gastric Subtype

Objective:To identify a high-risk group of patients with pancreatic ductal adenocarcinoma (PDAC), independently arising in the pancreas with intraductal papillary mucinous neoplasm (IPMN), using histopathologic subtypes. Background:Pathologic features of IPMN with distinct PDAC, including histopathologic subtypes of IPMN and PDAC phenotypes, have not been well characterized. Mucin expression patterns and the mutational status of GNAS and KRAS are useful to explore the relationship between these 2 lesion types. Methods:Clinicopathologic data of 179 resected IPMNs and 180 resected PDACs without IPMNs as a control group were reviewed. IPMNs were classified into 4 grades (low-grade, intermediate-grade, high-grade dysplasia, and an associated invasive carcinoma) and 4 subtypes (gastric, intestinal, pancreatobiliary, and oncocytic). The expression of MUC1, MUC2, MUC5AC, MUC6, and CDX2 was investigated by immunohistochemistry in IPMNs and PDACs with and without IPMNs. The mutational status of GNAS and KRAS was evaluated by cycle sequencing in PDACs and pre-/coexisting IPMNs. Results:Twenty-six synchronous or metachronous PDACs were identified in 20 patients (11.2%) with IPMNs. Occurrence of concomitant PDACs was more frequently observed in gastric-type IPMNs (18/110, 16.4%) compared with intestinal (1/49, 2.0%), pancreatobiliary (1/17, 5.9%), or oncocytic-type (0/3, 0%) (P = 0.047). Both PDACs with and without IPMNs were frequently positive for MUC1, MUC5AC, and MUC6 expression, as assessed by immunohistochemistry, but were negative for MUC2 and CDX2. The mucin-staining patterns were similar to those of invasive tubular adenocarcinoma arising from gastric-type IPMNs. Mutation of GNAS within codon 201 was not detected in PDACs and gastric-type IPMNs, whereas most of these exhibited KRAS mutations. However, the R201H GNAS mutation was detected in 1 intestinal-type IPMN with distinct PDAC. Conclusions:Mucin expression patterns demonstrate that PDAC without GNAS mutations of an aggressive phenotype frequently arise in the pancreas with benign gastric-type IPMN in the absence of GNAS mutations.