Paclitaxel is a microtubule-targeting antineoplastic drug widely used in human cancers. Even when tumors are initially responsive, progression of disease despite continued taxane therapy is all too common in the treatment of many of the most common epithelial cancers, including breast cancer. However, the mechanisms underlying paclitaxel resistance in cancer cells are not completely understood. Our hypothesis is that changes in the intrinsic (or mitochondrial) cell death pathway controlled by the BCL-2 family are key to the development of acquired paclitaxel resistance. Here we show that paclitaxel activates the mitochondrial apoptosis pathway, which can be blocked by BCL-2 overexpression. Treatment with ABT-737, a small-molecule BCL-2 antagonist, restores sensitivity to paclitaxel in BCL-2-overexpressing cells. To investigate the importance of changes in the intrinsic apoptotic pathway in the absence of enforced BCL-2 expression, we generated two independent breast cancer cell lines with acquired resistance to apoptosis induced by paclitaxel. In these lines, acquired resistance to paclitaxel is mediated either by increased antiapoptotic BCL-2 proteins or decreased proapoptotic BCL-2 proteins. In both cases, ABT-737 can engage the mitochondrial apoptosis pathway to restore sensitivity to paclitaxel to cell lines with acquired paclitaxel resistance. In summary, these findings suggest that alterations in the intrinsic apoptotic pathway controlled by BCL-2 protein family members may be crucial to causing paclitaxel resistance. Furthermore, our results suggest that combining smallmolecule BCL-2 antagonists with paclitaxel may offer benefit to patients with paclitaxel-resistant tumors, an oncologic problem of great prevalence. [Cancer Res 2008;68(19):7985-94]

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Taxanes exert their antitumor effect via stabilizing microtubule dynamics and initiating G2/M arrest in cancer cells followed by apoptotic cell death. However, the signaling pathways that connect paclitaxel-induced microtubule perturbation to mitochondrial outer membrane permeabilization (MOMP) and cytochrome c release are not well characterized. Here we demonstrate that in breast cancer cells paclitaxel induces a novel displacement mechanism: prodeath BH3-only proteins Bmf and Puma competitively displace prodeath BH3-only protein Bim from anti-apoptotic proteins to activate Bax and Bak and commit the cell to apoptotic death. Bim and either Puma or Bmf are required for paclitaxel toxicity. While prior mechanisms of apoptosis induced by taxol have focused on changes in Bim levels, we find that an increase is not required for paclitaxel killing of breast cancer cells. Rather, competitive displacement of Bim from anti-apoptotic proteins is the key step committing the cell to death. This novel mechanism suggests the potential utility of novel therapies targeted at altering BH3-only protein heterodimerization.

Like many proteins, function and abundance of Bcl-2 family proteins are influenced by posttranslational modifications. These modifications include phosphorylation, proteolytic cleavage, ubiquitination, and proteosomal degradation. These modifications, depending on cellular context and the proteins involved, can result either in a promotion of inhibition of apoptosis. Many of these modifications are governed by the activity of enzymes. As modulation of enzymatic activity can be achieved fairly efficiently using small molecules, understanding the effects of posttranslational modifications may allow for the therapeutic inhibition or promotion of apoptosis.