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Frank Cuttitta, Ph.D.
Identification of Autocrine/Paracrine Growth Regulators in Human Cancers
The main focus of this project is to define the potential autocrine/paracrine mechanisms regulating the growth of human tumors and utilize these as rational targets for early detection and intervention of malignant disease. We have developed several investigative strategies to achieve this goal. Our initial effort was accomplished by acclimating tumor cell lines to grow in a protein-free/peptide-free medium (R0), thereby forcing the cell to express maximum survival capabilities in a nutrient-poor environment. Under these conditions we have been able to establish long-term growth (1 to 6 years) from a variety of epithelial cancer lines including those of lung, breast, colon, ovary, pancreas, prostate, and neuroblastoma/glioblastoma lineage. Biochemical analysis of the conditioned media from R0 cells revealed the presence of protein/peptides such as gastrin-releasing peptide (GRP), insulin-like growth factors (IGF-I/IGF-II), transforming growth factor-alpha (TGF-alpha), adrenomedullin (AM), and transferrin (Tf), which are established mitogens of tumor growth. Since R0 base medium contains no exogenous protein/peptides, the only source of such products was for the R0-adapted cells themselves. These conditions may mirror steps in carcinogenesis, which involve clonal expansion of the transformed cell. Thus by identifying the growth factors and their respective receptor systems expressed during R0 adaptation of tumor cell lines, we have been able to generate appropriate molecular and immunological reagents to evaluate their involvement during normal to malignant conversion in pathological specimens. In addition, since disruption of ligand/receptor binding results in growth cessation, these factors make potential targets to intervene in the carcinogenesis pathway by blocking the promotion event.
During the past several years we have focused our research on the peptide hormone adrenomedullin and evaluated its role in carcinogenesis, embryogenesis, wound repair, inflammation, and diabetes. AM was initially isolated and characterized from a human pheochromocytoma (adrenal tumor) shown to activate platelet adenylyl cyclase and induce hypotension when injected intravenously into experimental animals. We have found that AM and its respective receptor, AM-R, are highly expressed in a variety of human tumor cell lines (cancers of the lung, breast, colon, brain, ovary, prostate, and skin). This peptide was shown to mediate autocrine proliferation of the tumor cell (in vitro/in vivo), and its action could be blocked by an anti-AM monoclonal antibody in a dose-dependent manner, causing growth cessation. Similarly, we have shown, using both molecular and immunohistochemical techniques, that AM is upregulated during embryogenesis and that the peptide plays an important role in the implantation process. At the site of the ectoplacental cone (area of attachment where fetal trophoblasts invade maternal tissue, forming the decidual interface and thereby mimicking events associated with tumor metastasis), AM and AM-R were found to be highly expressed. Very recently, several laboratories have shown that homozygous knockouts of the AM gene result in a lethal deletion for embryogenesis. To circumvent the dilemma, we have begun construction on inducible knockout mice using a CRE/Lox technology with the intention of targeting the lung (CC10 promoter) for our initial study. If successful with this project, we will be able to study AM influence on lung development and, if not a lethal deletion for the adult mouse, determine what effect AM plays in pulmonary carcinogenesis.
We have also shown that AM functions as an antimicrobial peptide causing the lysis of both bacterial and fungal pathogens. Endotoxin-activated macrophages are the main source of AM expression in the immune response pathway, and plasma AM levels rise dramatically during sepsis. Very early in our investigation of AM, we demonstrated that AM is produced in the F cell of the pancreatic islet (cell that makes pancreatic peptide, PP) and that AM blocks glucose-induced insulin release from the beta cell. In collaboration with the United States Department of Agriculture (USDA), we have found that intraperitoneal injections of our anti-AM monoclonal antibody could lower the blood glucose levels of genetically engineered diabetic/obese rats (SKR/corpulent) which approached concentrations of normal controls. Additionally, with the USDA group, we have shown that endotoxin can enhance AM expression in the pancreatic islet and alter glucose metabolism in experimental cattle with superimposed parasitic infections, causing an augmentation in this phenomenon. Finally, in collaboration with the Uniformed Services University of the Health Sciences (USUHS), we have shown that AM expression in wound repair is elevated at sites of neovascularization and that topical application of this peptide can dampen drug-induced suppression of wound repair in dexamethasone-treated rats.
Our collective studies have identified potential targets for the early detection and intervention of malignant disease and offer a rational approach in the investigative avenues to define new biological markers of neoplasm expression.
Among our collaborators are Sam Bhathena and Ted Elsasser, U.S. Department of Agriculture; Nadya Tarasova and Lino Tessarollo, Advanced Bioscience Laboratories, Inc.; and Stefanie Vogel, Uniformed Services University of the Health Sciences.
This page was last updated on 5/13/2011.