Abstract
HSP90 inhibitors are currently undergoing clinical evaluation in combination with antimitotic drugs in non-small cell lung cancer (NSCLC), but little is known about the cellular effects of this novel drug combination. Therefore, we investigated the molecular mechanism of action of IPI-504 (retaspimycin HCl), a potent and selective inhibitor of HSP90, in combination with the microtubule targeting agent (MTA) docetaxel, in preclinical models of NSCLC. We identified a subset of NSCLC cell lines in which these drugs act in synergy to enhance cell death. Xenograft models of NSCLC demonstrated tumor growth inhibition, and in some cases, regression in response to combination treatment. Treatment with IPI-504 enhanced the antimitotic effects of docetaxel leading to the hypothesis that the mitotic checkpoint is required for the response to drug combination. Supporting this hypothesis, overriding the checkpoint with an Aurora kinase inhibitor diminished the cell death synergy of IPI-504 and docetaxel. To investigate the molecular basis of synergy, an unbiased stable isotope labeling by amino acids in cell culture (SILAC) proteomic approach was employed. Several mitotic regulators, including components of the ubiquitin ligase, anaphase promoting complex (APC/C), were specifically down-regulated in response to combination treatment. Loss of APC/C by RNAi sensitized cells to docetaxel and enhanced its antimitotic effects. Treatment with a PLK1 inhibitor (BI2536) also sensitized cells to IPI-504, indicating that combination effects may be broadly applicable to other classes of mitotic inhibitors. Our data provide a preclinical rationale for testing the combination of IPI-504 and docetaxel in NSCLC.
Highlights
The mitotic, or spindle assembly checkpoint helps maintain genomic integrity by preventing the missegregation of chromosomes
To identify non-small cell lung cancer (NSCLC) cell lines demonstrating in vivo sensitivity to the combination of IPI-504 and docetaxel, mice bearing xenograft tumors (H1993, A549, H522 and H292) were treated with vehicle, 50 mg/kg IPI-504 alone, 5 or 15 mg/kg docetaxel alone, or a combination of 50 mg/kg IPI-504 and 5 or 15 mg/kg docetaxel
Single-agent activity was observed upon treatment with docetaxel, resulting in growth inhibition compared to vehicle in H1993, A549 and H292 xenografts (56–69%) (Fig. 1A–C)
Summary
The mitotic, or spindle assembly checkpoint helps maintain genomic integrity by preventing the missegregation of chromosomes. A highly orchestrated surveillance system composed of numerous proteins detects unattached kinetochores, or lack of proper tension across the mitotic spindle, triggering the so-called ‘‘checkpoint response’’, which leads to mitotic arrest. Normal cell division requires successful passage through the mitotic checkpoint. Failure to satisfy checkpoint requirements within a relatively short timeframe (1–2 days) can result in aneuploidy, mitotic catastrophe, or mitotic slippage followed by a variety of cell fates including cell death, senescence, or endoreduplication [1]. While the mechanisms by which prolonged mitosis leads to cell death are unclear, a role for the anti-apoptotic BCL2 family members has been reported [2]. Cyclin-cyclin dependent kinase (CDK) proteins phosphorylate BCL2 family members including BCL2, BCL-XL, and MCL1. Functional redundancy is likely to exist among the BCL2 family members in mediating the cell death response to prolonged mitosis
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