Research Type:
 Minimally Invasive Surgery
Pancreatic
Gastrointestinal Epithelial Biology
Clinical Effectiveness
Clinical Research
Pancreatic Research Laboratory
Principal Investigators:
Andrew L. Warshaw, M.D.
Carlos Fernández-del Castillo, M.D.
Sarah P. Thayer, M.D., Ph.D.
Group Members:
Denise Long, MD
Nancy Neyhard
Amy Stirman
The laboratory has been active since 1973, and from 1989 on has had, at any given time, two or three research fellows as well as a research technician or technologist. Its focus has been pancreatic disease, in particular, pathogenesis of acute pancreatitis and pancreatic carcinogenesis. Research fellows typically spend two years in the lab, and in addition to carrying their own research projects, collaborate with research projects of the other fellows in the lab, and occasionally have been involved in clinical studies as well.
Current projects include:
The Role of Matrix Metalloproteinase in Neutrophil Migration During Lung Injury in Acute Necrotizing Pancreatitis:
Leukocyte-endothelial interaction is a key factor in the pathogenesis of pulmonary injury seen in acute pancreatitis. This interaction is dependent on adhesion molecules that allow for rolling and sticking of neutrophils. The final step in the migration of the neutrophil through the endothelial barrier depends on proteolytic degradation of the basal membrane. This requires upregulation of matrix metalloproteinase, and can be potentially arrested with MMP inhibitors.
Use of Protease and Complement Inhibitors in the Treatment of Acute Pancreatitis and the Prevention of its Systemic Complications:
We are currently investigating nafamostat mesilate which is a novel, highly potent protease and complement inhibitor. Utilizing a model of acute pancreatitis first described in our laboratory, we are investigating this product with different dosing schedules and routes of administration. We are able to quantify trypsinogen activation using a unique assay that measures trypsinogen activation peptides. Our laboratory was the first to describe the use of this assay in the study of experimental pancreatitis, and has extensive experience with its application. The study endpoints are lung injury, peritoneal permeability and survival.
Development of a Rat and Mouse Model of Pancreatic Ductal Adenocarcinoma:
This was described in our laboratory three years ago, and is currently undergoing further characterization, including the development of a transplantable tumor and cell line. The model has morphologic and molecular characteristics that are very similar to the carcinoma seen in humans, and has the potential to contribute to its better understanding and treatment.
Evaluation of Strategies to Prevent Tumor Implantation in Pancreatic Cancer:
Using a mouse model of liver metastases from pancreatic cancer, we are currently testing matrix metalloproteinase inhibitors and other antiangiogenic therapies to prevent implantation and increase survival.
Telomerase Activity in Pancreatic Cancer:
Utilizing fresh tissue samples of both pancreatic cancer and premalignant pancreatic lesions (such as mucinous cystadenomas and intraductal papillary mucinous tumors) we are investigating the role of telomerase in pancreatic carcinogenesis.
Near-Infrared Confocal Microscopy for Evaluation of Pancreatic Disease:
In collaboration with the Wellman laboratory of photomedicine, we are exploring the use of near-infrared confocal microscopy both in vivo and ex vivo in normal pancreas, pancreatitis, and pancreatic cancer. This may prove to be a unique tool for the evaluation of microcirculation and angioarchitecture of both inflammatory and malignant pancreatic disease.
Abnormal regulation of developmental pathways and its role in the genesis of pancreatic cancer:
Cancer cells subvert existing signaling and developmental pathways in order to survive, proliferate and metastasize. However, these pathways can also be used as possible novel treatment avenues. The focus of this investigator is to determine the molecular mechanisms that may contribute to pancreatic cancer formation. Specifically we are investigating the role of abnormal expression of a developmental signaling factor, Sonic hedgehog (Shh), in the genesis of pancreatic cancer. The Shh pathway has been shown to be one of the most fundamental signal transduction pathways in embryonic development. Its down regulation plays a key and early role in normal pancreatic organogensis. However, abnormal expression of the hedgehog pathway have been implicated in other tumor types in which uncontrolled activation of the Shh signaling pathways has lead to the malignant transformation of normal human epithelium. Our preliminary data reveals that Shh is abnormally expressed in high levels in preneoplastic ductal epithelium, as well as in adenocarcinoma; furthermore, this abnormal expression appears to be directing the morphologic features that are seen in preneoplastic as well as in invasive cancer. Shh also upregulates other protooncogenes such as c-kit, which may contribute to abnormal proliferative signals, found in pancreatic neoplasia. This line of investigation will begin to allow us to analyze the role of Shh in the development of pancreatic adenocarcinoma. This will allow better understanding of the molecular determinants behind pancreatic cancer and empower us to develop better treatments and diagnostic modalities.
Characterizing the role of Sonic hedgehog's mis-expression in pancreatic cancer:
The role of Shh appears to be directing a foregut (gastric, intestinal) metaplasia of both the epithelium and mesenchyme within the adult human pancreas. This metaplasia appears to be responsible for the morphologic features seen in pancreatic cancer. Misexpression of Shh in adult human pancreata like in the transgenic mouse model, in which Shh is driven by the pancreatic-specific promoter PDX1, causes development of intestinal characteristics within the pancreas. In response to Shh, the epithelium changes from a normal cuboidal to a columnar form; and organize into tubular complexes that were PAS-positive for basic mucin, typical for an intestinal epithelium as well as pancreatic neoplasia. Human pancreatic cancer is characterized not only by epithelial changes but also by an extensive desmoplatic reaction within the mesenchyme. This extensive desmoplastic reaction within the pancreatic mesenchyme expresses alpha smooth muscle actin, which is found in intestinal mesenchyme. Thus, Shh overexpression appears to direct the epithelial and mesenchymal changes seen in pancreatic cancer.
Transgenic mouse models:
To determine if Shh mis-expression is sufficient to drive pancreatic cancer formation. First generation transgenic mice in which mis-expression in the pancreas Shh is driven by a pancreatic specific promoter, PDX-1. These mice develop very abnormal pancreata that form epithelial and mesenchymal changes which resemble, morphologically and histologically, the changes seen in human pre-neoplastic lesions. These mice will be further characterized in order to determined if they will develop pancreatic cancer. Second generation transgenics will next be created in which Shh will be driven by a conditional promoter allowing regulated activation of Shh specifically in the adult mouse pancreas.
Preclinical investigation into the effects of Shh pathway agonist and antagonists on the biologic behavior of pancreatic cancer:
To determine if inhibition using small molecule antagonist of this pathway will have any significant impact on the biologic behavior of pancreatic cancer. In vitro assays of biologic behavior include cell viability, growth fraction, alteration in cell cycle, and the activation of apoptotic pathways. Finally, the malignant potential of pancreatic cells will be tested in vivo using SCID mice.
A. Cell viability assay-Alteration in cell number in response to inhibition of Shh will be determined by microtitration assay (MTT). If the estimated cell number in response to Hh pathway inhibition is decreased, the etiologies are either cellular death or arrest. This method allows us to rapidly titrate the appropriate time course and dose of antagonist.
B. Cell cycle arrest-Reduction in the number of cells may be secondary to cell cycle arrest in response to Hh signaling. Alteration in cell cycle will be evaluated by flow cytometry analysis. Determination of growth fraction in tissue culture explants will be determined by immunohistochemistry using antibodies directed to Ki-67 and anti-PCNA.
C. Apoptosis-Reduction in cell number could also be the result of the activation of programmed cell death pathways. The measure of apoptosis in pancreatic tissue and cell cultures will be determined by morphology and by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay.
D. Malignant potential-Initial therapeutic studies with Hh molecule will be tested in an orthotopic in vivo model of human pancreatic cancer in which human pancreatic cancer is injected into the peritoneum of SCID mice which are then subject to treatment with Shh agonist and antagonist. Experimental end points are mortality and tumor burden.
Investigate the role of NF-kB in activation of the Sonic hedgehog gene:
Shh is activated early in response to inflammation. Inflammatory states have been linked causally to many cancers including pancreatic cancer. The pro-inflammatory mediator NF-kB appears to upregulate Shh expression. In order to investigate the role of NF-kB we use a recombinant adenoviral vector, which overexpressed IKK beta a known activator of NF-kB. This system allows us to study the mechanism of Shh activation.
Characterizing c-kit over-expression in Pancreatic cancer:
Shh overexpression appears to drive the abnormal expression of c-kit within the neoplastic pancreas. c-kit is a protooncogene that encodes a receptor tryrosine kinase, which has been found to be an important etiologic agent in the transformation of other solid tumors such as GIST. Here we investigate the relationship between Shh expression and c-kit as well as the effect of c-kit over-expression in pancreatic tumor progression.
Preclinical investigation into the effects of c-kit pathway inhibition with STI-571:
To determine if inhibition of the c-kit, tyrosine kinase pathway, will have any significant impact on the biologic behavior of pancreatic cancer. In vitro assays of biologic behavior include cell viability, growth fraction, alteration in cell cycle, and the activation of apoptotic pathways. Finally, the malignant potential of pancreatic cells will be tested in vivo using SCID mice.
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