Norihiro Sato

Biomarker Discovery from Pancreatic Cancer Secretome Using a Differential Proteomic Approach

Quantitative proteomics can be used as a screening tool for identification of differentially expressed proteins as potential biomarkers for cancers. Candidate biomarkers from such studies can subsequently be tested using other techniques for use in early detection of cancers. Here we demonstrate the use of stable isotope labeling with amino acids in cell culture (SILAC) method to compare the secreted proteins (secretome) from pancreatic cancer-derived cells with that from non-neoplastic pancreatic ductal cells. We identified 145 differentially secreted proteins (>1.5-fold change), several of which were previously reported as either up-regulated (e.g. cathepsin D, macrophage colony stimulation factor, and fibronectin receptor) or down-regulated (e.g. profilin 1 and IGFBP-7) proteins in pancreatic cancer, confirming the validity of our approach. In addition, we identified several proteins that have not been correlated previously with pancreatic cancer including perlecan (HSPG2), CD9 antigen, fibronectin receptor (integrin β1), and a novel cytokine designated as predicted osteoblast protein (FAM3C). The differential expression of a subset of these novel proteins was validated by Western blot analysis. In addition, overexpression of several proteins not described previously to be elevated in human pancreatic cancer (CD9, perlecan, SDF4, apoE, and fibronectin receptor) was confirmed by immunohistochemical labeling using pancreatic cancer tissue microarrays suggesting that these could be further pursued as potential biomarkers. Lastly the protein expression data from SILAC were compared with mRNA expression data obtained using gene expression microarrays for the two cell lines (Panc1 and human pancreatic duct epithelial), and a correlation coefficient (r) of 0.28 was obtained, confirming previously reported poor associations between RNA and protein expression studies.

Exploration of global gene expression patterns in pancreatic adenocarcinoma using cDNA microarrays

Pancreatic cancer is the fifth leading cause of cancer death in the United States. We used cDNA microarrays to analyze global gene expression patterns in 14 pancreatic cancer cell lines, 17 resected infiltrating pancreatic cancer tissues, and 5 samples of normal pancreas to identify genes that are differentially expressed in pancreatic cancer. We found more than 400 cDNAs corresponding to genes that were differentially expressed in the pancreatic cancer tissues and cell lines as compared to normal pancreas. These genes that tended to be expressed at higher levels in pancreatic cancers were associated with a variety of processes, including cell-cell and cell-matrix interactions, cytoskeletal remodeling, proteolytic activity, and Ca(++) homeostasis. Two prominent clusters of genes were related to the high rates of cellular proliferation in pancreatic cancer cell lines and the host desmoplastic response in the resected pancreatic cancer tissues. Of 149 genes identified as more highly expressed in the pancreatic cancers compared with normal pancreas, 103 genes have not been previously reported in association with pancreatic cancer. The expression patterns of 14 of these highly expressed genes were validated by either immunohistochemistry or reverse transcriptase-polymerase chain reaction as being expressed in pancreatic cancer. The overexpression of one gene in particular, 14-3-3 sigma, was found to be associated with aberrant hypomethylation in the majority of pancreatic cancers analyzed. The genes and expressed sequence tags presented in this study provide clues to the pathobiology of pancreatic cancer and implicate a large number of potentially new molecular markers for the detection and treatment of pancreatic cancer.

Peritumoral Fibroblast SPARC Expression and Patient Outcome With Resectable Pancreatic Adenocarcinoma

PURPOSE SPARC (secreted protein acidic and rich in cysteine) is a protein involved in cell matrix interactions, wound repair, and cell migration, and has been reported to inhibit cancer growth. SPARC undergoes epigenetic silencing in many pancreatic cancers, but stromal fibroblasts adjacent to infiltrating pancreatic adenocarcinomas frequently express SPARC. We evaluated the prognostic significance of tumor and peritumoral SPARC expression in patients with pancreatic adenocarcinoma. PATIENTS AND METHODS The expression patterns of SPARC were characterized by immunohistochemistry in 299 primary pancreatic ductal adenocarcinoma resection specimens from patients who underwent pancreaticoduodenectomy at Johns Hopkins Hospital (Baltimore, MD) between 1998 and 2003. Kaplan-Meier analysis and Cox proportional hazards regression modeling were used to assess the mortality risk associated with the presence or absence of tumor SPARC and peritumoral SPARC status. RESULTS By Kaplan-Meier analysis, patients whose pancreatic cancer stromal fibroblasts expressed SPARC (median survival, 15 months) had a significantly worse prognosis than patients whose tumor stroma did not express SPARC (median survival, 30 months; log-rank P < .001). In contrast, the expression of SPARC in pancreatic cancer cells was not associated with prognosis (log-rank P = .13). Controlling for other prognostic factors (tumor size, positive lymph nodes, margin status, tumor grade, and age), the relative hazard for patients whose stroma expressed SPARC compared with those whose stroma did not was 1.89 (95% CI, 1.31 to 2.74); the expression of SPARC in pancreatic cancer cells remained unrelated to prognosis (relative hazard, 1.02; 95% CI, 0.73 to 1.42). CONCLUSION The expression of SPARC by peritumoral fibroblasts portends a poorer prognosis for patients with pancreatic cancer.

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.

SPARC/osteonectin is a frequent target for aberrant methylation in pancreatic adenocarcinoma and a mediator of tumor-stromal interactions.

Deregulated expression of SPARC/osteonectin, a secreted glycoprotein with multiple biological functions, has been associated with the progression of various cancers. Using microarrays, we previously identified SPARC as one of the genes induced by treatment with a DNA methylation inhibitor in pancreatic cancer cells. We therefore analysed the expression pattern and methylation status of the SPARC gene in pancreatic cancer. Gene expression profiling by oligonucleotide microarray and reverse transcription–PCR analyses demonstrated that SPARC mRNA was expressed in non-neoplastic pancreatic ductal epithelial cells, but was not expressed in a majority of pancreatic cancer cell lines. The loss of SPARC expression was associated with aberrant hypermethylation of its CpG island. Immunohistochemical labeling revealed that the SPARC protein was overexpressed in the stromal fibroblasts immediately adjacent to the neoplastic epithelium in primary pancreatic cancers, but rarely expressed in the cancers themselves. Primary fibroblasts derived from pancreatic cancer strongly expressed SPARC mRNA and secreted SPARC protein into the conditioned media, and treatment of pancreatic cancer cells with exogenous SPARC resulted in growth suppression. SPARC expression in fibroblasts from noncancerous pancreatic tissue was augmented by coculture with pancreatic cancer cells. These findings suggest that SPARC is a frequent target for aberrant methylation in pancreatic cancer and that SPARC expression in fibroblasts adjacent to pancreatic cancer cells is regulated through tumor–stromal interactions.

STK11/LKB1 Peutz-Jeghers Gene Inactivation in Intraductal Papillary-Mucinous Neoplasms of the Pancreas

Despite the growing awareness of intraductal papillary-mucinous neoplasms (IPMNs) of the pancreas among clinicians, the molecular features of IPMNs have not been well characterized. Previous reports suggest that inactivation of the STK11/LKB1, a tumor-suppressor gene responsible for Peutz-Jeghers syndrome (PJS), plays a role in the pathogenesis of gastrointestinal hamartomas as well as several cancers, including pancreatic adenocarcinoma. Using polymerase chain reaction amplification of five microsatellite markers from the 19p13.3 region harboring the STK11/LKB1 gene, we analyzed DNA from 22 IPMNs for loss of heterozygosity (LOH). LOH at 19p13.3 was identified in 2 of 2 (100%) IPMNs from patients with PJS and 5 of 20 (25%) from patients lacking features of PJS (7 of 22, 32% overall). Sequencing analysis of the STK11/LKB1 gene in these IPMNs with LOH revealed a germline mutation in one IPMN that arose in a patient with PJS and a somatic mutation in 1 of the 20 sporadic IPMNs. None of the 22 IPMNs showed hypermethylation of the STK11/LKB1 gene. These results suggest that the STK11/LKB1 gene is involved in the pathogenesis of some IPMNs.

Serum Macrophage Inhibitory Cytokine 1 as a Marker of Pancreatic and Other Periampullary Cancers

Purpose: Patients with pancreatic ductal adenocarcinoma usually present with advanced-stage disease and a dismal prognosis. One effective strategy likely to improve the morbidity and mortality from pancreatic cancer would be the identification of accurate, noninvasive diagnostic markers that would enable earlier diagnosis of symptomatic patients and earlier detection of cancer in asymptomatic individuals at high risk for developing pancreatic cancer. In this study, we evaluated serum macrophage inhibitory cytokine-1 (MIC-1) as a marker of pancreatic cancer. Experimental Design: MIC-1 expression in primary pancreatic cancers, intraductal papillary mucinous neoplasms, and pancreatic cancer cell lines was determined using the National Center for Biotechnology Information serial analysis of gene expression database, oligonucleotide microarrays analysis, in situ hybridization, and immunohistochemistry. Serum MIC-1 levels were determined by ELISA in 80 patients with pancreatic adenocarcinomas, in 30 patients with ampullary and cholangiocellular carcinomas, in 42 patients with benign pancreatic tumors, in 76 patients with chronic pancreatitis, and in 97 healthy control subjects. The diagnostic performance of serum MIC-1 as a marker of pancreatic cancer was compared with that of serum CA19–9. Results: Oligonucleotide microarray and serial analysis of gene expression data demonstrated that MIC-1 RNA levels were higher in primary pancreatic cancers, intraductal papillary mucinous neoplasms, and pancreatic cancer cell lines than in nonneoplastic pancreatic ductal epithelium. MIC-1 expression was localized to the malignant epithelium in pancreatic adenocarcinomas by in situ hybridization. MIC-1 protein was expressed in 14 of 16 primary pancreatic adenocarcinomas (88%) by immunohistochemistry and was also expressed in some pancreata affected by pancreatitis but not in normal pancreas. Serum MIC-1 levels were significantly higher in patients with pancreatic ductal adenocarcinoma (mean ± SD, 2428 ± 2324 pg/ml) and in patients with ampullary and cholangiocellular carcinomas (2123 ± 2387 pg/ml) than in those with benign pancreatic neoplasms (940 ± 469 pg/ml), chronic pancreatitis (1364 ± 1236 pg/ml), or in healthy controls (546 ± 262 pg/ml). An elevated serum MIC-1 (defined as 2 SD above the mean for healthy controls) performed as well as CA19–9 (area under the receiver operating characteristic curve, 0.81 and 0.77, respectively), and the combination of MIC-1 and CA19–9 significantly improved diagnostic accuracy (P < 0.05; area under the receiver operating characteristic curve, 0.87; sensitivity, 70%; specificity, 85%). Conclusion: Serum MIC-1 measurement can aid in the diagnosis of pancreatic adenocarcinoma.

Aberrant methylation of preproenkephalin and p16 genes in pancreatic intraepithelial neoplasia and pancreatic ductal adenocarcinoma.

Pancreatic intraductal neoplasia (PanIN) is thought to be the precursor to infiltrating pancreatic ductal adenocarcinoma. We have previously shown that the preproenkephalin (ppENK) and p16 genes are aberrantly methylated in pancreatic adenocarcinoma. In this study we define the methylation status of the ppENK and p16 genes in various grades of PanINs. One hundred seventy-four samples (28 nonneoplastic pancreatic epithelia, 7 reactive epithelia, 29 PanIN-1A, 48 PanIN-1B, 27 PanIN-2, 14 PanIN-3, 15 invasive ductal adenocarcinomas, and 6 miscellaneous pancreatic neoplasms) were microdissected from 29 formalin-fixed paraffin-embedded surgically resected pancreata, and were analyzed by methylation-specific polymerase chain reaction. Fourteen of 15 (93.3%) invasive pancreatic ductal adenocarcinomas showed methylation of the ppENK gene and 4 of 15 (26.7%) showed methylation of the p16 gene. Nonneoplastic pancreatic epithelia did not harbor methylation of either gene. The prevalence of methylation of the ppENK gene increased significantly with increasing PanIN grade. A similar nonsignificant trend was noted for p16 methylation. Aberrant methylation of the ppENK gene was found in 7.7% of PanIN-1A, 7.3% of PanIN-1B, 22.7% of PanIN-2, and 46.2% of PanIN-3. Aberrant methylation of the p16 gene was found in 12% of PanIN-1A, 2.6% of PanIN-1B, 4.5% of PanIN-2, and 21.4% of PanIN-3. All but one of the PanINs from the 14 pancreata without pancreatic carcinoma was unmethylated with respect to either the p16 or ppENK gene. Our results suggest that methylation-related inactivation of the ppENK and p16 genes is an intermediate or late event during pancreatic carcinogenesis. Because aberrant methylation of ppENK or p16 was more often detected in similar grade PanINs from patients with pancreatic carcinoma than in those with other pancreatic diseases, it may be a useful indicator of the potential malignancy of epithelial cells of the pancreas.

DNA methylation alterations in the pancreatic juice of patients with suspected pancreatic disease

Molecular markers of pancreatic neoplasia could aid in the evaluation of visible pancreatic lesions and indicate neoplasia invisible to imaging. We evaluated methylation-specific PCR (MSP) assays that detect aberrantly methylated DNA for their use as markers of pancreatic neoplasia. Methylation analysis was done on pancreatic juice collected endoscopically or surgically from 155 individuals with suspected pancreatic disease: 56 patients had pancreatic ductal adenocarcinoma, 17 had intraductal papillary mucinous neoplasms, 26 had symptomatic chronic pancreatitis, 12 controls lacked evidence of pancreatic disease, and 44 were asymptomatic individuals at increased risk of developing familial pancreatic cancer undergoing screening for pancreatic neoplasia. Pancreatic juice DNA was analyzed for promoter methylation using conventional MSP assays for 17 genes. For six genes, pancreatic juice methylation was quantified using real-time quantitative MSP (QMSP; Cyclin D2, FOXE1, NPTX2, ppENK, p16, and TFPI2). Quantifying pancreatic juice methylation using QMSP with a cutoff of >1% methylated DNA could better predict pancreatic cancer than detecting methylation using conventional MSP. In the endoscopic group, 9 of 11 patients with pancreatic cancer, but none of 64 individuals without neoplasia had > or =1% methylation for two or more of the best five QMSP assays (82% sensitivity and 100% specificity; P < 0.0001). The prevalence of pancreatic juice methylation in patients with chronic pancreatitis was less than in patients with pancreatic cancer but higher than in controls and similar to high-risk individuals. The detection and quantification of aberrantly methylated DNA in pancreatic juice is a promising approach to the diagnosis of pancreatic cancer.

Gene Expression Profiling Identifies Genes Associated with Invasive Intraductal Papillary Mucinous Neoplasms of the Pancreas

The molecular pathology of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas has not been well characterized, and there are no reliable markers to predict the presence of an associated invasive carcinoma in IPMNs. Using oligonucleotide microarrays, we performed a large-scale gene expression profiling of 12 IPMNs with or without an associated invasive carcinoma. A subset of genes identified was validated for the gene expression patterns in a large panel of IPMNs by reverse-transcription polymerase chain reaction and/or immunohistochemistry. A total of 673 transcripts were identified as expressed at significantly higher levels (P < 0.05 and at fivefold or greater) in IPMNs relative to normal pancreatic ductal epithelial samples. Of interest, many of the genes identified as overexpressed in IPMNs have also been previously reported to be highly expressed in infiltrating ductal adenocarcinoma of the pancreas. By analyzing genes overexpressed selectively in IPMNs with an associated invasive carcinoma (n = 7), we also identified a panel of genes potentially associated with the invasive phenotype of the neoplasms. Immunohistochemical validation revealed that claudin 4, CXCR4, S100A4, and mesothelin were expressed at significantly high frequency in invasive IPMNs than in noninvasive IPMNs. Notably, the expression of at least two of the four proteins was observed in 73% of 22 invasive IPMNs but in none of 16 noninvasive IPMNs (P < 0.0001). Our findings suggest that preoperative assessment of gene expression profiles may be able to differentiate invasive from noninvasive IPMNs.