Identifying molecular targets for cancer therapy

Abstract: Within cancer research a molecular target is a specific molecular attribute in a cancer cell or tumor tissue that may play a role in promoting disease progression. There are many of these molecular attributes present in cancer biology and they represent events for therapeutic intervention. However, the major challenge for cancer therapy is selecting the right time to exploit a molecular target, or targets, while destroying cancer cells, and preserving normal cells. Therefore, critical to the success of any cancer therapy, is utilizing new technology and experimental approaches to gain a basic knowledge of the molecular mechanisms causing the disease and promoting its progression, and also developing and utilizing new therapeutic strategies. The aim of this thesis was to identify novel molecular targets for cancer therapy, and to develop new therapeutic strategies in a widely studied mouse model of melanoma. The first project was a discovery based proteomics study to identify differential protein expression during tumor progression. Since proteins are the most commonly targeted molecule for therapeutics, a comparative proteome analysis was performed on B16-F10 derived tumors in C57BL/6 mice at days 3, 5, 7, and 10. Hierarchical clustering of 44 protein spots (p<0.01) revealed a clear switch in expression of these proteins between the day 5 and the day 7 tumors. Additionally, a trend analysis showed 6 predominant kinetic paths of protein expression as the tumor progressed. Proteins involved in glycolysis, inflammation, wounding, superoxide metabolism, and chemotaxis increased during tumorigenesis. From day 3 to day 7 VEGF and active cathepsin D were induced 7-fold and 4-fold respectively. Proteins involved in electron transport, protein folding, blood coagulation, and transport decreased during tumorigenesis. Identified proteins from this kinetic proteome analysis elucidated tumorigenic processes during tumor progression and therapeutic targets. The second project in this thesis was the development and the successful use of a novel biological therapy. Phage display technologies have been widely used for the identification of tumor targeting peptides and antibodies, and phages are known to be highly immunogenic. Two tumor specific phages were developed to test if localizing phage particles to a tumor epitope would generate an anti-phage immune response, and as a bystander effect, destroy the tumor. Tumor therapy using the tumor specific phages was evaluated using the B16-F10 mouse model of melanoma. Treatment with tumor specific phages was superior to treatment with non-specific phages. We reveal a novel biological cancer treatment demonstrating that tumor specific phages can promote regression of established tumors. The third project in this thesis was a focused study for identifying therapeutic targets by comparing protein expression of pro-angiogenic and pro-tumorigenic molecules in normal vs. cancerous cells. Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine with pro-inflammatory, pro-angiogenic and pro-tumorigenic properties, was discovered to be over-expressed in the B16-F10 melanoma cells and not in syngeneic melanocytes. The molecular mechanisms underlying the role of MIF in tumorigenesis and angiogenesis are not well understood, and to address these roles, an interfering MIF RNA (iMIF) was stably introduced into the B16-F10 mouse melanoma cell line. When iMIF cells were subcutaneously injected into C57BL/6 mice, tumor establishment was significantly delayed and there was a marked absence of intratumoral vasculature in iMIF tumors relative to controls. In microarray analyses of iMIF and control melanoma cell lines thrombospondin 1 (TSP-1) mRNA expression was found to be up-regulated 55-fold (p<0.05) in the iMIF cells, and a 2-fold increase in TSP-1 protein levels was observed in iMIF cell culture supernatants. These results strongly suggest that reduced vasculature and consequently the delayed tumor establishment in iMIF melanomas are linked to the up-regulation of the anti-angiogenic TSP-1. They further define a novel function of MIF as a regulator of TSP-1 and, thus, angiogenesis in a mouse melanoma model.