Mechanism of cancer evading metronomic chemotherapy and action of Archazolid as an anti-metastatic drug

In the present study the mechanisms leading to acquired chemoresistance, as well as new treatment strategies implying the prevention evading of tumor cells were addressed. Resistance formation is one of the major hurdles in cancer therapy. Metronomic antiangiogenic treatment of xenografted prostate cancer tumors in mice with cyclophosphamide (CPA) results in the appearance of resistant tumors. To investigate the complex molecular changes occurring during resistance formation, a comprehensive gene expression analysis of the resistant tumors in vivo was performed. A multitude of differentially expressed genes, e.g. PAS domain containing protein 1 (PASD1), annexin A3 (ANXA3), neurotensin (NTS) or plasminogen activator tissue (PLAT), were observed, when comparing resistant to in vivo passaged tumor samples. Moreover, tumor cells from in vivo and in vitro conditions showed a significant difference in target gene expression. For clarification of the mechanisms leading to the survival of tumor cells during maintained anti-angiogenic CPA therapy the differentially expressed genes were assigned to functional pathways like: axon guidance, steroid biosynthesis and complement and coagulation cascades. As blood flow might play a crucial role during maintained anti-angiogenic therapy, further analysis was focused on the genes grouped in complement and coagulation cascades. pregulation of anti-coagulatory ANXA3 and PLAT and downregulation of SERPIN A1 and other SERPIN-family members was shown by qPCR analysis. In contrast coagulation factor F3 was upregulated, accompanied by the expression of an altered gene product. Taken together, a potential role of anticoagulation as a resistance mechanism for anti-angiogenic CPA therapy could be described. Furthermore, the role of archazolid, a novel myxobacterial V-ATPase inhibitor in cancer treatment and in particular its action on the secreted cellular proteome was evaluated. As extracellular protein secretion may have an impact on invasive properties of tumor cells, the changes of the secretome profile of highly migratory urinary bladder carcinoma cells upon archazolid treatment were analyzed. An induced secretion of prometastatic lysosomal proteins such as the cathepsin family was observed. Interestingly, intracellular cathepsin B activity however strongly decreases and mature cathepsin B protein diminishes. It could be shown that archazolid inhibits the mannose-6-phosphate receptor mediated trafficking of procathepsin B from the trans-Golgi network to prelysosomal compartments, leading to an impaired cathepsin B maturation process. This results in an unnatural secretion of the inactive proenzyme and a dramatic decrease in intracellular cathepsin B activity. Importantly, also in vivo an archazolid induced reduction of cathepsin B activity was proven and archazolid treatment resulted in a reduced formation of distant metastases in the lungs. In summary these results indicate that archazolid in addition to its known anti-migratory properties might exert an anti-metastatic effect by reducing the activity of pro metastatic proteases like cathepsin B.