Faculty Bibliography

Pancreatic cancer is the fourth leading cause of cancer-related death in the United States, with a 5-year survival rate of less than 4%. Effective early detection and screening are currently not available, and tumors are typically diagnosed at a late stage, frequently after metastasis. Existing clinical markers of pancreatic cancer lack specificity, as they are also found in inflammatory diseases of the pancreas and biliary tract. In the work described here, naturally occurring glycoproteins were enriched by using lectin affinity chromatography and then further resolved by nonporous reversed-phase chromatography. Glycoprotein microarrays were then printed and probed with a variety of lectins to screen glycosylation patterns in sera from normal, chronic pancreatitis, and pancreatic cancer patients. Ten normal, 8 chronic pancreatitis, and 6 pancreatic cancer sera were investigated. Data from the glycoprotein microarrays were analyzed using bioinformatics approaches including principal component analysis (PCA) and hierarchical clustering (HC). Both normal and chronic pancreatitis sera were found to cluster close together, although in two distinct groups, whereas pancreatic cancer sera were significantly different from the other two groups. Both sialylation and fucosylation increased as a function of cancer on several proteins including Hemopexin, Kininogen-1, Antithrombin-III, and Haptoglobin-related protein, whereas decreased sialylation was detected on plasma protease C1 inhibitor. Target alterations on glycosylations were verified by lectin blotting experiments and peptide mapping experiments using microLC-ESI-TOF. These altered glycan structures may have utility for the differential diagnosis of pancreatic cancer and chronic pancreatitis and identify critical differences between biological samples from patients with different clinical conditions.

OBJECTIVES: Serum biomarkers for early diagnosis of pancreatic adenocarcinoma are not currently available. We recently observed elevated expression of CEACAM1 in pancreatic adenocarcinomas and sought to determine whether serum CEACAM1 levels were elevated in pancreatic cancer patients. METHODS: CEACAM1 messenger RNA levels were measured in pancreatic tissue samples using quantitative reverse transcription-polymerase chain reaction. CEACAM1 was localized by immunohistochemistry in adenocarcinomas and in pancreatic intraductal neoplasia lesions. CEACAM1 serum levels were assessed by a double determinant enzyme-linked immunosorbent assay and compared with serum levels of CA19-9. RESULTS: CEACAM1 had higher expression levels in pancreatic adenocarcinomas compared with noncancerous pancreas (P < 0.0001) and was localized to neoplastic cells (95% (45/47) of adenocarcinomas and 85% (17/20) of pancreatic intraductal neoplasia 3 lesions. CEACAM1 was expressed in the sera of 91% (74/81) of pancreatic cancer patients, 24% (15/61) of normal patients, and 66% (35/53) of patients with chronic pancreatitis, with a sensitivity and specificity superior to CA19-9. The combination of CEACAM1 and CA19-9 had significantly higher diagnostic accuracy than CA19-9. CONCLUSIONS: CEACAM1 is expressed in pancreatic adenocarcinoma, and serum levels of CEACAM1 serve as a useful indicator for the presence of pancreatic cancer. Additional validation studies on the use of serum CEACAM1 as a diagnostic marker in pancreatic cancer are warranted.

Carbohydrate post-translational modifications on proteins are important determinants of protein function in both normal and disease biology. We have developed a method to allow the efficient, multiplexed study of glycans on individual proteins from complex mixtures, using antibody microarray capture of multiple proteins followed by detection with lectins or glycan-binding antibodies. Chemical derivatization of the glycans on the spotted antibodies prevented lectin binding to those glycans. Multiple lectins could be used as detection probes, each targeting different glycan groups, to build up lectin binding profiles of captured proteins. By profiling both protein and glycan variation in multiple samples using parallel sandwich and glycan-detection assays, we found cancer-associated glycan alteration on the proteins MUC1 and CEA in the serum of pancreatic cancer patients. Antibody arrays for glycan detection are highly effective for profiling variation in specific glycans on multiple proteins and should be useful in diverse areas of glycobiology research.

The current study investigated adrenomedullin as a potential autocrine regulator of pancreatic cancer cell function. Adrenomedullin was localized in the neoplastic epithelium of 90% (43 of 48) of human pancreatic adenocarcinomas analyzed by immunohistochemistry and was expressed by 100% (8 of 8) of pancreatic cancer cell lines analyzed by reverse transcription-PCR. Pancreatic cancer cell lines also secreted adrenomedullin into the culture medium as determined by ELISA (5 of 5). Exogenous adrenomedullin treatment of Panc-1, BxPC3, and MPanc96 cells in vitro stimulated cell proliferation, invasion, and nuclear factor kappaB activity, indicating the ability of the cells to respond to adrenomedullin. Treatment of the cell cultures with an adrenomedullin antagonist inhibited basal levels of proliferation and nuclear factor kappaB activity, supporting the autocrine function of this molecule. Furthermore, increasing adrenomedullin levels by gene transfer to Panc-1 cells increased, whereas adrenomedullin small hairpin RNA silencing in MPanc96 cells inhibited tumor growth and metastasis in vivo. Adrenomedullin is able to act through at least two different receptors, adrenomedullin receptor (ADMR) and calcitonin receptor-like receptor (CRLR). Reverse transcription-PCR and Western blotting indicated that pancreatic cancer cells expressed only ADMR but not CRLR. In contrast, cells found in the tumor microenvironment, primary human pancreatic stellate and endothelial (HUVEC) cells, expressed both ADMR and CRLR. Small hairpin RNA silencing of ADMR in pancreatic cancer cells blocked adrenomedullin-induced growth and invasion, indicating that this receptor is involved in the autocrine actions of adrenomedullin. These data indicate that adrenomedullin acting via ADMR increases the aggressiveness of pancreatic cancer cells and suggests that these molecules may be useful therapeutic targets.

Glycoproteins play important roles in various biological processes including intracellular transport, cell recognition, and cell-cell interactions. The change of the cellular glycosylation profile may have profound effects on cellular homeostasis and malignancy. Therefore, we have developed a sensitive screening approach for the comprehensive analysis of N-glycans and glycosylation sites on human serum proteins. Using this approach, N-linked glycopeptides were extracted by double lectin affinity chromatography. The glycans were enzymatically cleaved from the peptides and then profiled using capillary hydrophilic interaction liquid chromatography coupled online with ESI-TOF MS. The structures of the separated glycans were determined by MALDI quadrupole ion-trap TOF mass spectrometry in both positive and negative modes. The glycosylation sites were elucidated by sequencing of PNGase F modified glycopeptides using nanoRP-LC-ESI-MS/MS. Alterations of glycosylation were analyzed by comparing oligosaccharide expression of serum glycoproteins at different disease stages. The efficiency of this method was demonstrated by the analysis of pancreatic cancer serum compared to normal serum. Ninety-two individual glycosylation sites and 202 glycan peaks with 105 unique carbohydrate structures were identified from approximately 25 mug glycopeptides. Forty-four oligosaccharides were found to be distinct in the pancreatic cancer serum. Increased branching of N-linked oligosaccharides and increased fucosylation and sialylation were observed in samples from patients with pancreatic cancer. The methodology described in this study may elucidate novel, cancer-specific oligosaccharides and glycosylation sites, some of which may have utility as useful biomarkers of cancer.

Emerging evidence has suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells within a tumor, termed cancer stem cells. Although data have been provided to support this theory in human blood, brain, and breast cancers, the identity of pancreatic cancer stem cells has not been determined. Using a xenograft model in which primary human pancreatic adenocarcinomas were grown in immunocompromised mice, we identified a highly tumorigenic subpopulation of pancreatic cancer cells expressing the cell surface markers CD44, CD24, and epithelial-specific antigen (ESA). Pancreatic cancer cells with the CD44(+)CD24(+)ESA(+) phenotype (0.2-0.8% of pancreatic cancer cells) had a 100-fold increased tumorigenic potential compared with nontumorigenic cancer cells, with 50% of animals injected with as few as 100 CD44(+)CD24(+)ESA(+) cells forming tumors that were histologically indistinguishable from the human tumors from which they originated. The enhanced ability of CD44(+)CD24(+)ESA(+) pancreatic cancer cells to form tumors was confirmed in an orthotopic pancreatic tail injection model. The CD44(+)CD24(+)ESA(+) pancreatic cancer cells showed the stem cell properties of self-renewal, the ability to produce differentiated progeny, and increased expression of the developmental signaling molecule sonic hedgehog. Identification of pancreatic cancer stem cells and further elucidation of the signaling pathways that regulate their growth and survival may provide novel therapeutic approaches to treat pancreatic cancer, which is notoriously resistant to standard chemotherapy and radiation.

Total pancreatectomy has been used to treat both benign and malignant disease of the pancreas, but its use has been limited by concerns about management of the a-pancreatic state with its attendant total endocrine and exocrine insufficiency. Here, we review the indications for total pancreatectomy, operative technique, and improvements in the postoperative management of patients. Total pancreatectomy remains a viable option in the treatment of intractable pain associated with chronic pancreatitis, multicentric or extensive neuroendocrine tumors, patients with familial pancreatic cancer with premalignant lesions, and in patients with intraductal papillary mucinous neoplasia with diffuse ductal involvement or invasive disease. Improvements in postoperative management include auto-islet cell transplantation, advances in insulin formulations, and the use of glucagon rescue therapy which allow much tighter control of blood glucose than previously possible. This markedly lessens the risk of life-threatening hypoglycemia and decreases the risk of long-term complications, resulting in improved quality of life for these patients.

To investigate the role of transforming growth factor (TGF)-beta family signaling in the adult pancreas, a transgenic mouse (E-dnSmad4) was created that expresses a dominant-negative Smad4 protein driven by a fragment of the elastase promoter. Although E-dnSmad4 mice have normal growth, pancreas weight, and pancreatic exocrine and ductal histology, beginning at 4-6 wk of age, E-dnSmad4 mice show an age-dependent increase in the size of islets. In parallel, an expanded population of replicating cells expressing the E-dnSmad4 transgene is found in the stroma between the enlarged islets and pancreatic ducts. Despite the marked enlargement, E-dnSmad4 islets contain normal ratios and spatial organization of endocrine cell subtypes and have normal glucose homeostasis. Replication of cells derived from primary duct cultures of wild-type mice, but not E-dnSmad4 mice, was inhibited by the addition of TGF-beta family proteins, demonstrating a cell-autonomous effect of the transgene. These data show that, in the adult pancreas, TGF-beta family signaling plays a role in islet size by regulating the growth of a pluripotent progenitor cell residing in the periductal stroma of the pancreas.

Protein glycosylation has been implicated in key biological processes including immunological recognition, cellular adhesion, protein folding, and signaling as well as disease progression. Although several methods are available to assess glycosylation of protein structures, none of them is able to screen complex biological samples at a global as well as an individual scale. A novel strategy presented here uses an all-liquid phase enrichment and prefractionation methodology coupled to glycoprotein microarray technology using a multiple lectin-based, biotin-streptavidin detection scheme. Selective detection of glycan structures was made possible by employing multiple lectins to screen glycoprotein standards as well as serum samples from normal subjects or patients with chronic pancreatitis or pancreatic cancer. Interestingly, in some instances, a greater degree of glycosylation was seen in proteins that were underexpressed based on the reversed-phase chromatogram alone. Studies with standard proteins established the limits of detection to be in the 2.5-5-fmol range. Studies on serum samples showed differences in glycosylation patterns, particularly with respect to sialylation, mannosylation, and fucosylation, in normal, pancreatitis, and cancer sera. By coupling glycoprotein enrichment and fractionation with a microarray platform, we have shown that naturally occurring glycoproteins from human serum can be screened and characterized for different glycan structures, thereby allowing one to do comparative studies that monitor individual glycosylation changes within a glycoproteome representing different biological states. This approach may be useful to identify potential biomarkers in cancer.