The V-ATPase inhibitor archazolid

Fighting metastasis is a major challenge in cancer therapy and novel therapeutic targets and drugs are highly appreciated. Resistance of invasive cells to anoikis, a particular type of apoptosis induced by loss of cell-extracellular matrix (ECM) contact, is a major prerequisite for their metastatic spread. Inducing anoikis in metastatic cancer cells is therefore a promising therapeutic approach. The vacuolar H+-ATPase (V-ATPase), a proton pump located at the membrane of acidic organelles, has recently come to focus as an anti-metastatic cancer target. As V-ATPase inhibitors have shown to prevent invasion of tumor cells and are able to induce apoptosis we proposed that V-ATPase inhibition induces anoikis related pathways in invasive cancer cells. In this study the V-ATPase inhibitor archazolid A was used to investigate the mechanism of anoikis induction in various metastatic cancer cells (T24, MDA-MB-231, 4T1, 5637). Therefore, cells were forced to stay in a detached status to mimic loss of cell-ECM engagement following treatment with archazolid. Indeed, anoikis induction by archazolid was characterized by decreased expression of the caspase-8 inhibitor c-FLIP and caspase-8 activation, thus triggering the extrinsic apoptotic pathway. Interestingly, active integrin β1, which is known to play a major role in anoikis induction and resistance, is reduced on the cell surface of archazolid treated cells. Furthermore, a diminished phosphorylation of the integrin downstream target focal adhesion kinase could be demonstrated. The intrinsic apoptotic pathway was initiated by the pro-apoptotic protein BIM, increasing early after treatment. BIM activates cytochrome C release from the mitochondria consequently leading to cell death and is described as one major inducer of anoikis in non-malignant and anoikis sensitive cancer cells. Of note, we observed that archazolid also induces mechanisms opposing anoikis such as proteasomal degradation of BIM mediated by the pro-survival kinases ERK, c-Src and especially Akt at later time points. Moreover, induction of reactive oxygen species (ROS) influences BIM removal as well, as moderate levels of ROS have second messenger properties amplifying cell survival signals. Thus, to antagonize these anoikis escape strategies a combination of archazolid with proteasome or ROS inhibitors amplified cancer cell death synergistically. Most importantly, intravenous injection of archazolid treated 4T1-Luc2 mouse breast cancer cells in BALB/cByJRj mice resulted in reduced lung metastases in vivo. To summarize this work we propose archazolid as a very potent drug in inducing anoikis pathways in metastatic cancer cells even though having learned that detachment together with treatment triggers multiple resistance mechanisms opposing cell death. Hence, V-ATPase inhibition is not only an interesting option to reduce cancer metastasis but also to better understand anoikis resistance and to find choices to fight against it