Paclitaxel is used to treat breast cancers, but the mechanisms by which it induces apoptosis are poorly understood. Consequently, we have studied the role of the FoxO transcription factors in determining cellular response to paclitaxel. Western blotting revealed that in a panel of nine breast cancer cell lines expression of FoxO1a and FoxO3a correlated with the expression of the pro-apoptotic FoxO target Bim, which was associated with paclitaxel-induced apoptosis. In MCF-7 cells, which were paclitaxel-sensitive, the already high basal levels of FoxO3a and Bim protein increased dramatically after drug treatment, as did Bim mRNA, which correlated with apoptosis induction. This was not observed in MDA-231 cells, which expressed low levels of FoxOs and Bim. Gene reporter experiments demonstrated that in MCF-7 cells maximal induction of Bim promoter was dependent on a FoxO binding site, suggesting that FoxO3a is responsible for the transcriptional up-regulation of Bim. Gene silencing experiments showed that small interference RNA (siRNA) specific for FoxO3a reduced the levels of FoxO3a and Bim protein as well as inhibited apoptosis in paclitaxel-treated MCF-7 cells. Furthermore, siRNA specific for Bim reduced the levels of Bim protein and inhibited apoptosis in paclitaxel-treated MCF-7 cells. This is the first demonstration that up-regulation of FoxO3a by paclitaxel can result in increased levels of Bim mRNA and protein, which can be a direct cause of apoptosis in breast cancer cells.Breast cancer is one of the most common malignancies affecting women in the western world and arises following the accumulation of a series of somatic changes that serve to increase the rate of cellular proliferation and/or reduce the levels of apoptosis. These changes are often found to involve deregulation of key signal transduction pathways. Signal transduction pathways within the cell transmit the extracellular signals to transcription factors, resulting in changes in gene expression. These changes in gene expression are often cell typespecific and lead to cell growth, differentiation, or apoptosis, thereby regulating normal tissue structure and function. One key signal transduction pathway highly conserved in eukaryotes is the phosphatidylinositol 3-kinase (PI3K) 1 pathway (1), which is stimulated by a number of growth factors, including insulin and insulin-like growth factor 1 (1). Once a receptor tyrosine kinase has become activated by binding a specific ligand, it becomes autophosphorylated and binds PI3K heterodimers either directly, or indirectly via insulin receptor substrate (IRS) adaptor proteins (1-3). The interaction between the p85 regulatory subunit of PI3K and the receptor brings the catalytic domain (p110 subunit) of PI3K into contact with the plasma membrane, where it is able to phosphorylate its targets. Alternatively, PI3K can be juxtaposed to the plasma membrane via interactions with activated RAS family members. PI3K phosphorylates phosphatidylinositol (4,5)-diphosphate at the 3 position, resulting in the formatio...

Inhibitors of the mitogen-activated protein kinases (MAPK), BRAF, and MAP-ERK kinase (MEK) induce tumor regression in the majority of patients with BRAF-mutant metastatic melanoma. The clinical benefit of MAPK inhibitors is restricted by the development of acquired resistance with half of those who benefit having progressed by 6 to 7 months and long-term responders uncommon. There remains no agreed treatment strategy on disease progression in these patients. Without published evidence, fears of accelerated disease progression on inhibitor withdrawal have led to the continuation of drugs beyond formal disease progression. We now show that treatment with MAPK inhibitors beyond disease progression can provide significant clinical benefit, and the withdrawal of these inhibitors led to a marked increase in the rate of disease progression in two patients. We also show that MAPK inhibitors retain partial activity in acquired resistant melanoma by examining drug-resistant clones generated to dabrafenib, trametinib, or the combination of these drugs. All resistant sublines displayed a markedly slower rate of proliferation when exposed to MAPK inhibitors, and this coincided with a reduction in MAPK signaling, decrease in bromodeoxyuridine incorporation, and S-phase inhibition. This cytostatic effect was also associated with diminished levels of cyclin D1 and p-pRb. Two short-term melanoma cultures generated from resistant tumor biopsies also responded to MAPK inhibition, with comparable inhibitory changes in proliferation and MAPK signaling. These data provide a rationale for the continuation of BRAF and MEK inhibitors after disease progression and support the development of clinical trials to examine this strategy.