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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.

Objectives: Aberrant expression of several microRNAs (miRs) has been reported in various neoplasms including intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. MicroRNA-196a (miR-196a) is up-regulated in Barrett esophagus (characterized by intestinal metaplasia) and in colorectal cancer; this relationship between intestinal characteristics and miR-196a might also be applicable to intestinal-type IPMNs. The aim of this study was to evaluate whether intestinal-type IPMNs can be discriminated from non-intestinal-type IPMNs by the expression level of miR-196a in tissue and pancreatic juice samples., Methods: Thirty-seven formalin-fixed paraffin-embedded tissue samples (including 3 of normal pancreatic ducts) and 36 pancreatic juice samples were obtained. The expression level of miR-196a measured by quantitative reverse transcription-polymerase chain reaction assays was compared between intestinal-type and non-intestinal-type IPMNs., Results: MicroRNA-196a expression in intestinal-type IPMN tissue samples (n = 18) was significantly higher than that of non-intestinal-type IPMNs (n = 16) (P < 0.001). Similarly, miR-196a expression in pancreatic juice samples of intestinal-type IPMNs (n = 6) was significantly higher than that of non-intestinal-type IPMNs (n = 30) (P = 0.008), and the sensitivity and specificity for prediction of intestinal-type IPMNs using pancreatic juice samples were both 83%., Conclusions: Elevated expression of miR-196a in pancreatic juice samples is predictive of intestinal-type IPMNs.

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., (Copyright © 2011. Published by Mosby, Inc.)

Gemcitabine (GEM) is the standard treatment for advanced/metastatic pancreatic cancer. However, there is a substantial subset of patients in whom the efficacy of GEM, when used as a single agent, is inadequate. Recently, the 5-fluorouracil (5-FU) prodrugs capecitabine and S-1 have been used as an alternative, either alone or in combination with GEM. The aim of the present study was to investigate the expression pattern of genes that render pancreatic cancer cells sensitive to GEM and 5-FU, and to identify markers for individualized chemotherapy, even in patients who have developed resistance. We investigated the correlation between the expression of genes associated with the metabolism of GEM and 5-FU, and sensitivity to these drugs in 15 human pancreatic cancer cell lines. We also established GEM- and 5-FU-resistant pancreatic cancer cell lines to investigate changes in the expression levels of these genes and the effects of one drug on cells resistant to the other. We found no correlation between pancreatic cancer cell sensitivity to either GEM- or 5-FU. GEM-resistant cells did not become resistant to 5-FU and vice versa. High expression of RRM1 (P=0.048) and TS x DPD (P=0.035) correlated significantly with sensitivity to GEM and 5-FU, respectively. 5-FU-resistant cells expressed significantly higher levels of TP than parental cells (P<0.05). In conclusion, pancreatic cancer cells showed no cross-resistance to GEM and 5-FU. Quantitative analyses of RRM1, TP, DPD and TS mRNA levels in pancreatic cancer cells may be useful for predicting their sensitivity to GEM and 5-FU.

A hypoxic microenvironment is a characteristic feature of pancreatic cancer, and induces the expressions of various genes involved in malignant behaviors. Insulin-induced gene 2 (Insig2) has recently been shown to be correlated with cellular invasion in colon cancer. However, there have been no reports regarding its expression in pancreatic cancer. In this study, we evaluated Insig2 mRNA expression and the biological function of Insig2 in pancreatic cancer. We measured Insig2 mRNA expression in cultured pancreatic cancer cell lines and invasive ductal carcinoma (IDC) cells, normal pancreatic epithelial cells, and pancreatic intraepithelial neoplasia cells obtained by laser-capture microdissection. We also investigated the effects of Insig2-targeting siRNAs on the cell proliferation and cell invasion of pancreatic cancer cell lines. All pancreatic cancer cell lines expressed Insig2 mRNA. The PANC-1 and MIA PaCa-2 pancreatic cancer cell lines showed >2-fold higher Insig2 mRNA expression levels under hypoxic conditions (1% O2) than under normoxic conditions (21% O2 ). Cell proliferation was significantly decreased in SUIT-2 cells and cell invasion was significantly decreased in SUIT-2, Capan-2, and CFPAC-1 cells after transfection of the Insig2-targeting siRNAs. In analyses of microdissected cells, cells from IDC tissues expressed significantly higher levels of Insig2 mRNA than normal pancreatic cells (P < 0.001) and pancreatic intraepithelial neoplasia cells (P = 0.082). In analyses of IDC cells, the levels of Insig2 mRNA expression were significantly higher in late-stage patients than in early-stage patients. The present data suggest that Insig2 is associated with the malignant potential of pancreatic cancer under hypoxic conditions., (© 2011 Japanese Cancer Association.)

The microRNA-17-92 cluster is an oncogene in human B cell lymphomas and lung cancers. Previous microRNA microarray data revealed that miR-17-5p, a member of the miR-17-92 cluster, is upregulated in pancreatic cancer. However, the involvement of miR-17-5p expression in pancreatic carcinogenesis has not well been studied. In the present study, we measured the miR-17-5p expression levels in pancreatic cancer cell lines, primary cultures of normal human pancreatic ductal cells, formalin-fixed paraffin-embedded (FFPE) tissue samples derived from 80 patients who underwent pancreatectomy for pancreatic cancer and microdissected cells (including normal ductal epithelial, pancreatic intraepithelial neoplasia-1B and invasive ductal carcinoma cells) by qRT-PCR. Furthermore, we investigated the effects of upregulation of miR-17-5p expression on the proliferation and invasion of pancreatic cancer cells. We found that pancreatic cancer cells expressed higher levels of miR-17-5p than primary cultured normal ductal cells. miR-17-5p was also overexpressed in pancreatic cancer in FFPE and microdissected samples. Furthermore, analysis of macrodissected FFPE samples revealed that high miR-17-5p expression was associated with a poor prognosis (p = 0.03). In addition, in vitro experiments revealed that SUIT-2 and KP-2 pancreatic cancer cells transfected with the miR-17-5p precursor showed significantly higher cell growth ratios than the corresponding control cells (p < 0.001 and p = 0.012, respectively), as well as significantly higher numbers of invading cells (p < 0.0001 for both). The present findings suggest that miR-17-5p plays important roles in pancreatic carcinogenesis and cancer progression, and is associated with a poor prognosis in pancreatic cancer.

Background and Aims: The standard palliative chemotherapy for pancreatic ductal adenocarcinoma (PDAC) is gemcitabine-based chemotherapy; however, PDAC still presents a major therapeutic challenge. The aims of this study were to investigate the expression pattern of genes involved in gemcitabine sensitivity in resected PDAC tissues and to determine correlations of gene expression with treatment outcome., Materials and Methods: We obtained formalin-fixed paraffin-embedded (FFPE) tissue samples from 70 patients with PDAC. Of the 70 patients, 40 received gemcitabine-based adjuvant chemotherapy (AC). We measured hENT1, dCK, CDA, RRM1, and RRM2 messenger RNA (mRNA) levels by quantitative real-time reverse transcription-polymerase chain reaction and determined the combined score (GEM score), based on the expression levels of hENT1, dCK, RRM1, and RRM2, to investigate the association with survival time. By determining the expression levels of these genes in endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) cytologic specimens, we investigated the feasibility of individualized chemotherapy., Results: High dCK (P = .0067), low RRM2 (P = .003), and high GEM score (P = .0003) groups had a significantly longer disease-free survival in the gemcitabine-treated group. A low GEM score (<2) was an independent predictive marker for poor outcome to gemcitabine-based AC as shown by multivariate analysis (P = .0081). Altered expression levels of these genes were distinguishable in microdissected neoplastic cells from EUS-FNA cytologic specimens., Conclusions: Quantitative analyses of hENT1, dCK, RRM1, and RRM2 mRNA levels using FFPE tissue samples and microdissected neoplastic cells from EUS-FNA cytologic specimens may be useful in predicting the gemcitabine sensitivity of patients with PDAC.

Background: LIM-only 4 (LMO4), a member of the LIM-only (LMO) subfamily of LIM domain-containing transcription factors, was initially reported to have an oncogenic role in breast cancer. We hypothesized that LMO4 may be related to pancreatic carcinogenesis as it is in breast carcinogenesis. If so, this could result in a better understanding of tumorigenesis in pancreatic cancer., Methods: We measured LMO4 mRNA levels in cultured cells, pancreatic bulk tissues and microdissected target cells (normal ductal cells; pancreatic intraepithelial neoplasia-1B [PanIN-1B] cells; PanIN-2 cells; invasive ductal carcinoma [IDC] cells; intraductal papillary-mucinous adenoma [IPMA] cells; IPM borderline [IPMB] cells; and invasive and non-invasive IPM carcinoma [IPMC]) by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR)., Results: 9 of 14 pancreatic cancer cell lines expressed higher levels of LMO4 mRNA than did the human pancreatic ductal epithelial cell line (HPDE). In bulk tissue samples, expression of LMO4 was higher in pancreatic carcinoma than in intraductal papillary-mucinous neoplasm (IPMN) or non-neoplastic pancreas (p < 0.0001 for both). We carried out microdissection-based analyses. IDC cells expressed significantly higher levels of LMO4 than did normal ductal epithelia or PanIN-1B cells (p < 0.001 for both) or PanIN-2 cells (p = 0.014). IPMC cells expressed significantly higher levels of LMO4 than did normal ductal epithelia (p < 0.001), IPMA (p < 0.001) and IPMB cells (p = 0.003)., Conclusion: Pancreatic carcinomas (both IDC and IPMC) expressed significantly higher levels of LMO4 mRNA than did normal ductal epithelia, PanIN-1B, PanIN-2, IPMA and IPMB. These results suggested that LMO4 is overexpressed at late stages in carcinogenesis of pancreatic cancer.

Controversy exists regarding the clinical significance of S100A2 in the progression of tumours. In pancreatic cancer, little is known about the role of S100A2. The aim of this study was to clarify the clinical significance of S100A2 expression in pancreatic carcinogenesis. We microdissected invasive ductal carcinoma (IDC) cells from 22 lesions, pancreatic intraepithelial neoplasia (PanIN) cells from five lesions, intraductal papillary mucinous neoplasm (IPMN) cells from 38 lesions, pancreatitis-affected epithelial (PAE) cells from 16 lesions, and normal ductal cells from 18 normal pancreatic tissues. S100A2 expression in 14 pancreatic cancer cell lines, microdissected cells and formalin-fixed paraffin-embedded (FFPE) samples was examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Microdissection analyses revealed that IDC cells expressed higher levels of S100A2 than did IPMN, PAE or normal cells (all comparisons, p < 0.007). Cell lines from metastatic sites expressed higher levels of S100A2 than those from primary sites. PanIN cells expressed higher levels of S100A2 than normal cells (p = 0.018). IDC cells associated with poorly differentiated adenocarcinoma expressed higher levels of S100A2 than did IDC cells without poorly differentiated adenocarcinoma (p = 0.006). Analyses of FFPE samples revealed that levels of S100A2 were higher in samples from patients who survived < 1000 days after surgery than in those from patients who survived > 1000 days (p = 0.043). Immunohistochemical analysis was consistent with qRT-PCR. S100A2 may be a marker of tumour progression or prognosis in pancreatic carcinogenesis and pancreatic cancer., (Copyright 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Despite evidence that Twist, a highly conserved basic helix-loop-helix transcription factor, is a novel oncogene, there are no reports describing Twist expression in pancreatic cancer. Intraductal papillary mucinous neoplasm (IPMN) and pancreatic intraepithelial neoplasia (PanIN) are precursor lesions of pancreatic cancer. To clarify involvement of Twist expression in pancreatic cancer, we used quantitative reverse transcription-polymerase chain reaction and examined Twist expression in pancreatic cancer, IPMN, and non-neoplastic pancreas using bulk tissues (11 cancers, 18 IPMNs, and 15 non-neoplastic pancreata), microdissected cells (cancer from 22 sections, IPMN from 19 sections, PanIN from 6 sections, and pancreatitis-affected epithelial cells from 14 sections), and pancreatic juice (16 from cancer, 28 from IPMN, and 17 from pancreatitis). Twist expression differed significantly between cancer and IPMN bulk tissues (p < 0.0001) but not between cancer and non-neoplastic tissues. Twist expressions differed significantly between microdissected cancer cells, IPMN cells, and pancreatitis-affected cells (all comparisons, p < 0.017). PanIN cells expressed significantly lower levels of Twist than did IDC cells (p = 0.016). Twist expression differed significantly between cancer and IPMN juice samples (p = 0.0002) but not between cancer and pancreatitis juice samples. Receiver operation characteristic curve analyses revealed that measurement of Twist was more useful for discriminating cancer from IPMN than from chronic pancreatitis (p = 0.009). Our results suggest that Twist is involved in tumor progression of pancreatic cancer and that measurement of Twist in pancreatic juice may be useful to differentiate pancreatic cancer from nonmalignant neoplasms such as IPMN., ((c) 2007 Wiley-Liss, Inc.)

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.