Faculty Bibliography

A unique approach of automating the integration of monolithic capillary HPLC-based protein separation and on-plate digestion for subsequent MALDI-MS analysis has been developed. All liquid-handling procedures were performed using a robotic module. This automated high-throughput method minimizes the amount of time and extensive labor required for traditional in-solution digestion followed by exhaustive sample cleanup and analysis. Also, precise positioning of the droplet from the capillary HPLC separation onto the MALDI plate allows for preconcentration effects of analytes for improved sensitivity. Proteins from primary esophageal Barrett's adenocarcinoma tissue were prefractionated by chromatofocusing and analyzed successfully by this automated configuration, obtaining rapid protein identifications through PMF and sequencing analyses with high sequence coverage. Additionally, intact protein molecular weight values were obtained as a means to further confirm protein identification and also to identify potential sequence modifications of proteins. This simple and rapid method is a highly versatile and robust approach for the analysis of complex proteomes.

A strategy is developed in this study for identifying sialylated glycoprotein markers in human cancer serum. This method consists of three steps: lectin affinity selection, a liquid separation and characterization of the glycoprotein markers using mass spectrometry. In this work, we use three different lectins (Wheat Germ Agglutinin, (WGA) Elderberry lectin,(SNA), Maackia amurensis lectin, (MAL)) to extract sialylated glycoproteins from normal and cancer serum. Twelve highly abundant proteins are depleted from the serum using an IgY-12 antibody column. The use of the different lectin columns allows one to monitor the distribution of alpha(2,3) and alpha(2,6) linkage type sialylation in cancer serum vs that in normal samples. Extracted glycoproteins are fractionated using NPS-RP-HPLC followed by SDS-PAGE. Target glycoproteins are characterized further using mass spectrometry to elucidate the carbohydrate structure and glycosylation site. We applied this approach to the analysis of sialylated glycoproteins in pancreatic cancer serum. Approximately 130 sialylated glycoproteins are identified using microLC-MS/MS. Sialylated plasma protease C1 inhibitor is identified to be down-regulated in cancer serum. Changes in glycosylation sites in cancer serum are also observed by glycopeptide mapping using microLC-ESI-TOF-MS where the N83 glycosylation of alpha1-antitrypsin is down regulated. In addition, the glycan structures of the altered proteins are assigned using MALDI-QIT-MS. This strategy offers the ability to quantitatively analyze changes in glycoprotein abundance and detect the extent of glycosylation alteration as well as the carbohydrate structure that correlate with cancer.

Pancreatic cancer is a difficult and unsolved surgical problem. It remains one of the top five causes of cancer-related deaths and has the lowest 5-year survival of any cancer, largely due to late diagnosis, low resection rates, and local recurrence. Clinical trials examining the optimal timing and delivery of adjuvant therapies for pancreatic cancer have yielded controversial results. Although most experts agree that the addition of chemotherapy has survival benefit in patients with resectable pancreatic cancer, there is no consensus regarding the optimal therapeutic agents, timing (neoadjuvant versus adjuvant), and the addition of radiation therapy to the treatment regimen. Multiple phase III trials are in progress in efforts to examine these issues. Additionally, exciting progress has been made with novel chemotherapeutic combinations, and alternative treatment modalities including interferon-alpha, immunotherapy, and pancreatic cancer stem cells. Given the high failure pattern after surgical resection, with more than half of patients developing locoregional recurrence, all patients undergoing pancreaticoduodenectomy are candidates for adjuvant therapy.

Human chromosome 1p35-p36 has long been suspected to harbor a tumor suppressor gene in pancreatic cancer and other tumors. We found that expression of rap1GAP, a gene located in this chromosomal region, is significantly down-regulated in pancreatic cancer. Only a small percentage of preneoplastic pancreatic intraductal neoplasia lesions lost rap1GAP expression, whereas loss of rap1GAP expression occurred in 60% of invasive pancreatic cancers, suggesting that rap1GAP contributes to pancreatic cancer progression. In vitro and in vivo studies showed that loss of rap1GAP promotes pancreatic cancer growth, survival, and invasion, and may function through modulation of integrin activity. Furthermore, we showed a high frequency of loss of heterozygosity of rap1GAP in pancreatic cancer. Collectively, our data identify rap1GAP as a putative tumor suppressor gene in pancreatic cancer.

Pancreatic cancer has a poor prognosis, in part due to lack of early detection. The identification of circulating tumor antigens or their related autoantibodies provides a means for early cancer diagnosis. We have used a proteomic approach to identify proteins that commonly induce a humoral response in pancreatic cancer. Proteins from a pancreatic adenocarcinoma cell line (Panc-1) were subjected to two-dimensional PAGE, followed by Western blot analysis in which individual sera were tested for autoantibodies. Sera from 36 newly diagnosed patients with pancreatic cancer, 18 patients with chronic pancreatitis and 15 healthy subjects were analyzed. Autoantibodies were detected against a protein identified by mass spectrometry as vimentin, in sera from 16/36 patients with pancreatic cancer (44.4%). Only one of 18 chronic pancreatitis patients and none of the healthy controls exhibited reactivity against this vimentin isoform. Interestingly, none of several other isoforms of vimentin detectable in 2-D gels exhibited reactivity with patient sera. Vimentin protein expression levels were investigated by comparing the integrated intensity of spots visualized in 2-D PAGE gels of various cancers. Pancreatic tumor tissues showed greater than a 3-fold higher expression of total vimentin protein than did the lung, colon, and ovarian tumors that were analyzed. The specific antigenic isoform was found at 5-10 fold higher levels. The detection of autoantibodies to this specific isoform of vimentin may have utility for the early diagnosis of pancreatic cancer.

PURPOSE: In the current study, we examined the functional significance and mechanism of action of S100P in pancreatic cancer cells. EXPERIMENTAL DESIGN: S100P levels were increased in Panc-1 cells, which do not express S100P, by transfection with an S100P cDNA and S100P levels were reduced in BxPC3 cells, which express high levels of S100P, by small interfering RNA gene silencing. Effects of these manipulations on cell proliferation, resistance to apoptotic insults, cell migration, and invasion were estimated in vitro using standard assays. The influences of S100P on tumor growth in vivo were studied using xenograft mouse models. To identify the mechanisms involved in these responses, coimmunoprecipitation studies were conducted with S100P with receptor for advanced glycation end products (RAGE) and the effects of inhibiting RAGE using an antagonistic peptide were analyzed. RESULTS: S100P levels correlated with the rates of cell proliferation, survival, migration, and invasion in both cell models in vitro. In vivo, increased S100P levels increased the growth of tumors in mice with s.c.-implanted Panc-1 cells and decreased S100P levels decreased tumor growth after orthotopic implantation of BxPC-3 cells. A direct interaction between S100P and RAGE was indicated by coimmunoprecipitation of these molecules from pancreatic cancer cells. A RAGE antagonist peptide inhibited this interaction and also inhibited the biological effects of S100P on these cells in vitro. CONCLUSIONS: These data suggest that S100P plays a major role in the aggressiveness of pancreatic cancer that is likely mediated by its ability to activate RAGE. Thus, interference with S100P may provide a novel approach for treatment of pancreatic cancer.

OBJECTIVES: Tissue desmoplasia occurs in a number of disease states, but its molecular basis is poorly understood. To determine which genes are overexpressed in cells contained within the desmoplastic stroma of pancreatic adenocarcinoma and chronic pancreatitis, we undertook genetic profiling of microdissected tissue samples of pancreatic adenocarcinoma, chronic pancreatitis, normal pancreas, and pancreatic cancer cell lines. We observed that samples of both pancreatic adenocarcinoma and chronic pancreatitis showed elevated expression of many shared genes compared with the normal pancreas. We hypothesized that these common genes likely important in stromal production and/or function could be identified using a strategy that involved comparisons between pancreatic adenocarcinoma, chronic pancreatitis, normal pancreas, and pancreatic cancer cell lines. METHODS: We performed oligonucleotide microarray analysis of 6800 different genes expressed in 10 samples of pancreatic adenocarcinoma, 5 samples of normal pancreas, 5 samples of chronic pancreatitis, and 7 pancreatic cancer cell lines. Microarray findings were validated with RT-PCR, and immunohistochemistry was used to verify protein localization to the stromal compartment of both pancreatic cancer and chronic pancreatitis. RESULTS: We employed a deductive comparison whereby genes expressed in the normal pancreas and pancreatic cancer cell lines were selectively eliminated from those expressed in common by pancreatic adenocarcinoma and chronic pancreatitis. This strategy identified 107 genes predicted to be expressed within cells of the stromal compartment of both pancreatic adenocarcinoma and chronic pancreatitis. CONCLUSIONS: These genes are likely important factors in epithelial-stromal signaling in pancreatic desmoplasia and may serve as diagnostic or therapeutic targets.

BACKGROUND: Raf-1 kinase inhibitory protein (RKIP) was recently identified as a physiologic endogenous inhibitor of the extracellular signal-regulated kinase (ERK) pathway. The expression and role of RKIP within the pancreas are unknown. METHODS: RKIP expression in normal pancreas and human insulinomas was examined by using paraffin-embedded sections. Co-localization of RKIP within islet cell subtypes was performed by using double immunofluorescence staining with antibodies directed toward RKIP and endocrine markers. To examine the role of RKIP in beta-cell proliferation, stable expression of sense (ss) and antisense (as) RKIP was established in HIT-T15 beta cells. The effect of RKIP on the ERK-signaling pathway in beta cells was determined by Western blotting with the use of phospho-specific antibodies directed against mitogen-activated protein kinase kinase (MEK) and ERK. The role of RKIP in beta-cell proliferation was assessed by using MTS assay and FACS analysis. RESULTS: RKIP was expressed only within pancreatic islet cells. Immunofluorescent double staining revealed that RKIP was expressed in most beta cells and a subset of pancreatic polypeptide-expressing cells. Based on the known function of RKIP, we hypothesized that RKIP expression would be downregulated in insulinomas: 8 of 9 human insulinomas demonstrated no RKIP staining, with decreased expression in 1 of 9 insulinomas. Studies using asRKIP and ssRKIP demonstrated that RKIP blocked activation of MEK and ERK by Raf-1 in beta cells. We also showed that RKIP inhibited beta-cell proliferation by altering cell cycle distribution, rather than by promoting apoptosis. CONCLUSIONS: RKIP is important in beta-cell proliferation, and its downregulation may play a role in islet neoplasia.

The identification of circulating tumor antigens or their related autoantibodies provides a means for early cancer diagnosis as well as leads for therapy. We have used a proteomic approach to identify proteins that commonly induce a humoral response in pancreatic cancer. Aliquots of solubilized proteins from a pancreatic cancer cell line (Panc-1) were subjected to two-dimensional PAGE, followed by Western blot analysis in which sera of individual patients were tested for primary antibodies. Sera from 36 newly diagnosed patients with pancreatic cancer, 18 patients with chronic pancreatitis, 33 patients with other cancers, and 15 healthy subjects were analyzed. Autoantibodies were detected against either one or two calreticulin isoforms identified by mass spectrometry in sera from 21 of 36 patients with pancreatic cancer. One of 18 chronic pancreatitis patients and 1 of 15 healthy controls demonstrated autoantibodies to calreticulin isoform 1; none demonstrated autoantibodies to isoform 2. None of the sera from patients with colon cancer exhibited reactivity against either of these two proteins. One of 14 sera from lung adenocarcinoma patients demonstrated autoantibodies to calreticulin isoform 1; 2 of 14 demonstrated autoantibodies to isoform 2. Immunohistochemical analysis of calreticulin in pancreatic/ampullary tumor tissue arrays using an isoform nonspecific antibody revealed diffuse and consistent cytoplasmic staining in the neoplastic epithelial cells of the pancreatic and ampullary adenocarcinomas. The detection of autoantibodies to calreticulin isoforms may have utility for the early diagnosis of pancreatic cancer.

Hypoxic conditions exist within pancreatic adenocarcinoma, yet pancreatic cancer cells survive and replicate within this environment. To understand the mechanisms involved in pancreatic cancer adaptation to hypoxia, we analyzed expression of a regulator of hypoxia-induced cell death, Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3). We found that BNIP3 was down-regulated in nine of nine pancreatic adenocarcinomas compared with normal pancreas despite the up-regulation of other hypoxia-inducible genes, including glucose transporter-1 and insulin-like growth factor-binding protein 3. Also, BNIP3 expression was undetectable even after hypoxia treatment in six of seven pancreatic cancer cell lines. The BNIP3 promoter, which was remarkably activated by hypoxia, is located within a CpG island. The methylation status of CpG dinucleotides within the BNIP3 promoter was analyzed after bisulfite treatment by sequencing and methylation-specific PCR. Hypermethylation of the BNIP3 promoter was observed in all BNIP3-negative pancreatic cancer cell lines and eight of 10 pancreatic adenocarcinoma samples. Treatment of BNIP3-negative pancreatic cancer cell lines with a DNA methylation inhibitor, 5-aza-2' deoxycytidine, restored hypoxia-induced BNIP3 expression. BNIP3 expression was also restored by introduction of a construct consisting of a full-length BNIP3 cDNA regulated by a cloned BNIP3 promoter. Restoration of BNIP3 expression rendered the pancreatic cancer cells notably more sensitive to hypoxia-induced cell death. In conclusion, down-regulation of BNIP3 by CpG methylation likely contributes to resistance to hypoxia-induced cell death in pancreatic cancer.