Activating mutations in KRAS and BRAF are found in more than 30% of all human tumours and 40% of melanoma, respectively, thus targeting this pathway could have broad therapeutic effects. Small molecule ATP-competitive RAF kinase inhibitors have potent antitumour effects on mutant BRAF(V600E) tumours but, in contrast to mitogen-activated protein kinase kinase (MEK) inhibitors, are not potent against RAS mutant tumour models, despite RAF functioning as a key effector downstream of RAS and upstream of MEK. Here we show that ATP-competitive RAF inhibitors have two opposing mechanisms of action depending on the cellular context. In BRAF(V600E) tumours, RAF inhibitors effectively block the mitogen-activated protein kinase (MAPK) signalling pathway and decrease tumour growth. Notably, in KRAS mutant and RAS/RAF wild-type tumours, RAF inhibitors activate the RAF-MEK-ERK pathway in a RAS-dependent manner, thus enhancing tumour growth in some xenograft models. Inhibitor binding activates wild-type RAF isoforms by inducing dimerization, membrane localization and interaction with RAS-GTP. These events occur independently of kinase inhibition and are, instead, linked to direct conformational effects of inhibitors on the RAF kinase domain. On the basis of these findings, we demonstrate that ATP-competitive kinase inhibitors can have opposing functions as inhibitors or activators of signalling pathways, depending on the cellular context. Furthermore, this work provides new insights into the therapeutic use of ATP-competitive RAF inhibitors.

Glycogen synthase kinase-3 (GSK-3)-alpha and -beta are closely related protein-serine kinases, which act as inhibitory components of Wnt signalling during embryonic development and cell proliferation in adult tissues. Insight into the physiological function of GSK-3 has emerged from genetic analysis in Drosophila, Dictyostelium and yeast. Here we show that disruption of the murine GSK-3beta gene results in embryonic lethality caused by severe liver degeneration during mid-gestation, a phenotype consistent with excessive tumour necrosis factor (TNF) toxicity, as observed in mice lacking genes involved in the activation of the transcription factor activation NF-kappaB. GSK-3beta-deficient embryos were rescued by inhibition of TNF using an anti-TNF-alpha antibody. Fibroblasts from GSK-3beta-deficient embryos were hypersensitive to TNF-alpha and showed reduced NF-kappaB function. Lithium treatment (which inhibits GSK-3; refs 8, 9) sensitized wild-type fibroblasts to TNF and inhibited transactivation of NF-kappaB. The early steps leading to NF-kappaB activation (degradation of I-kappaB and translocation of NF-kappaB to the nucleus) were unaffected by the loss of GSK-3beta, indicating that NF-kappaB is regulated by GSK-3beta at the level of the transcriptional complex. Thus, GSK-3beta facilitates NF-kappaB function.

Escherichia coli contains operons called "addiction modules," encoding toxin and antitoxin, which are responsible for growth arrest and cell death. Here, we demonstrate that MazF toxin encoded by "mazEF addiction module" is a sequence-specific (ACA) endoribonuclease functional only for single-stranded RNA. MazF works as a ribonuclease independent of ribosomes, and is, therefore, functionally distinct from RelE, another E. coli toxin, which assists mRNA cleavage at the A site on ribosomes. Upon induction, MazF cleaves whole cellular mRNAs to efficiently block protein synthesis. Purified MazF inhibited protein synthesis in both prokaryotic and eukaryotic cell-free systems. This inhibition was released by MazE, the labile antitoxin against MazF. Thus, MazF functions as a toxic endoribonuclease to interfere with the function of cellular mRNAs by cleaving them at specific sequences leading to rapid cell growth arrest and cell death. The role of such endoribonucleases may have broad implication in cell physiology under various growth conditions.

Epidermal growth factor receptor (EGFR) and HER3 each form heterodimers with HER2 and have independently been implicated as key coreceptors that drive HER2-amplified breast cancer. Some studies suggest a dominant role for EGFR, a notion of renewed interest given the development of dual HER2/EGFR small-molecule inhibitors. Other studies point to HER3 as the primary coreceptor. To clarify the relative contributions of EGFR and HER3 to HER2 signaling, we studied receptor knockdown via small interfering RNA technology across a panel of six HER2-overexpressing cell lines. Interestingly, HER3 was as critical as HER2 for maintaining cell proliferation in most cell lines, whereas EGFR was dispensable. Induction of HER3 knockdown in the HER2-overexpressing BT474M1 cell line was found to inhibit growth in three-dimensional culture and induce rapid tumor regression of in vivo xenografts. Furthermore, preferential phosphorylation of HER3, but not EGFR, was observed in HER2-amplified breast cancer tissues. Given these data suggesting HER3 as an important therapeutic target, we examined the activity of pertuzumab, a HER2 antibody that inhibits HER3 signaling by blocking ligand-induced HER2/HER3 heterodimerization. Pertuzumab inhibited ligand-dependent morphogenesis in three-dimensional culture and induced tumor regression in the heregulin-dependent MDA-MB-175 xenograft model. Importantly, these activities of pertuzumab were distinct from those of trastuzumab, a monoclonal antibody currently used for treatment of HER2-amplified breast cancer patients. Our data suggest that inhibition of HER3 may be more clinically relevant than inhibition of EGFR in HER2-amplified breast cancer and also suggest that adding pertuzumab to trastuzumab may augment therapeutic benefit by blocking HER2/ HER3 signaling.

Purpose: The pathways underlying basal-like breast cancer are poorly understood, and as yet, there is no approved targeted therapy for this disease. We investigated the role of mitogenactivated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitors as targeted therapies for basal-like breast cancer. Experimental Design: We used pharmacogenomic analysis of a large panel of breast cancer cell lines with detailed accompanying molecular information to identify molecular predictors of response to a potent and selective inhibitor of MEK and also to define molecular mechanisms underlying combined MEK and PI3K targeting in basal-like breast cancer. Hypotheses were confirmed by testing in multiple tumor xenograft models. Results: We found that basal-like breast cancer models have an activated RAS-like transcriptional program and show greater sensitivity to a selective inhibitor of MEK compared with models representative of other breast cancer subtypes.We also showed that loss of PTEN is a negative predictor of response to MEK inhibition, that treatment with a selective MEK inhibitor caused up-regulation of PI3K pathway signaling, and that dual blockade of both PI3K and MEK/extracellular signal^regulated kinase signaling synergized to potently impair the growth of basal-like breast cancer models in vitro and in vivo. Conclusions: Our studies suggest that single-agent MEK inhibition is a promising therapeutic modality for basal-like breast cancers with intact PTEN, and also provide a basis for rational combination of MEK and PI3K inhibitors in basal-like cancers with both intact and deleted PTEN.

Although Akt is known as a survival kinase, inhibitors of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway do not always induce substantial apoptosis. We show that silencing Akt1 alone, or any combination of Akt isoforms, can suppress the growth of tumors established from phosphatase and tensin homologue–null human cancer cells. Although these findings indicate that Akt is essential for tumor maintenance, most tumors eventually rebound. Akt knockdown or inactivation with small molecule inhibitors did not induce significant apoptosis but rather markedly increased autophagy. Further treatment with the lysosomotropic agent chloroquine caused accumulation of abnormal autophagolysosomes and reactive oxygen species, leading to accelerated cell death in vitro and complete tumor remission in vivo. Cell death was also promoted when Akt inhibition was combined with the vacuolar H+–adenosine triphosphatase inhibitor bafilomycin A1 or with cathepsin inhibition. These results suggest that blocking lysosomal degradation can be detrimental to cancer cell survival when autophagy is activated, providing rationale for a new therapeutic approach to enhancing the anticancer efficacy of PI3K–Akt pathway inhibition.

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Recent structural studies on calmodulin complexes with anthrax adenylyl cyclase and rat Ca2+-activated K+ channel have uncovered unexpected ways by which calmodulin interacts with target proteins.