ABSTRACT
A focus of contemporary cancer therapeutic development is the targeting of both the transformed cell and the supporting cellular microenvironment. Cell migration is a fundamental cellular behavior required for the complex interplay between multiple cell types necessary for tumor development. We therefore developed a novel retroviral-based screening technology in primary human endothelial cells to discover genes that control cell migration. We identified the receptor tyrosine kinase Axl as a novel regulator of endothelial cell haptotactic migration towards the matrix factor vitronectin. Using small interfering RNA-mediated silencing and overexpression of wild-type or mutated receptor proteins, we show that Axl is a key regulator of multiple angiogenic behaviors including endothelial cell migration, proliferation, and tube formation in vitro. Moreover, using sustained, retrovirally delivered short hairpin RNA (shRNA) Axl knockdown, we show that Axl is necessary for in vivo angiogenesis in a mouse model. Furthermore, we show that Axl is also required for human breast carcinoma cells to form a tumor in vivo. These findings indicate that Axl regulates processes vital for both neovascularization and tumorigenesis. Disruption of Axl signaling using a small-molecule inhibitor will hence simultaneously affect both the tumor and stromal cell compartments and thus represents a unique approach for cancer therapeutic development.
Subject(s)
Breast Neoplasms/enzymology , Cell Transformation, Neoplastic/metabolism , Oncogene Proteins/physiology , Receptor Protein-Tyrosine Kinases/physiology , Animals , Breast Neoplasms/blood supply , Cell Growth Processes/physiology , Cell Line, Tumor , Cell Movement/genetics , Cells, Cultured , Chemotaxis/drug effects , Chemotaxis/physiology , Coculture Techniques , Endothelial Cells/cytology , Endothelial Cells/enzymology , Humans , Mice , Mice, SCID , Neovascularization, Pathologic/enzymology , Neovascularization, Pathologic/genetics , Neovascularization, Physiologic/genetics , Neovascularization, Physiologic/physiology , Oncogene Proteins/antagonists & inhibitors , Oncogene Proteins/biosynthesis , Oncogene Proteins/genetics , Proto-Oncogene Proteins , RNA, Small Interfering/genetics , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor Protein-Tyrosine Kinases/genetics , Signal Transduction , Transfection , Transplantation, Heterologous , Vitronectin/pharmacology , Axl Receptor Tyrosine KinaseABSTRACT
Inteins are polypeptide sequences found in a small set of primarily bacterial proteins that promote the splicing of flanking pre-protein sequences to generate mature protein products. Inteins can be engineered in a "split and inverted" configuration such that the protein splicing product is a cyclic polypeptide consisting of the sequence linking two intein subdomains. We have engineered a split intein into a retroviral expression system to enable the intracellular delivery of a library of random cyclic peptides in human cells. Cyclization of peptides could be detected in cell lysates using mass spectrometry. A functional genetic screen to identify 5-amino acid-long cyclic peptides that block interleukin-4 mediated IgE class switching in B cells yielded 13 peptides that selectively inhibited germ line epsilon transcription. These results demonstrate the generation of cyclic peptide libraries in human cells and the power of functional screening to rapidly identify biologically active peptides.
