The Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) pathway participates in the control of many fundamental cellular processes including proliferation, survival, and differentiation. The pathway is deregulated in up to 30% of human cancers, often due to mutations in Ras and the B-Raf isoform. Raf-1 and B-Raf can form heterodimers, and this may be important for cellular transformation. Here, we have analyzed the biochemical and biological properties of Raf-1/B-Raf heterodimers. Isolated Raf-1/B-Raf heterodimers possessed a highly increased kinase activity compared to the respective homodimers or monomers. Heterodimers between wild-type Raf-1 and B-Raf mutants with low or no kinase activity still displayed elevated kinase activity, as did heterodimers between wild-type B-Raf and kinase-negative Raf-1. In contrast, heterodimers containing both kinase-negative Raf-1 and kinase-negative B-Raf were completely inactive, suggesting that the kinase activity of the heterodimer specifically originates from Raf and that either kinase-competent Raf isoform is sufficient to confer high catalytic activity to the heterodimer. In cell lines, Raf-1/B-Raf heterodimers were found at low levels. Heterodimerization was enhanced by 14-3-3 proteins and by mitogens independently of ERK. However, ERK-induced phosphorylation of B-Raf on T753 promoted the disassembly of Raf heterodimers, and the mutation of T753 prolonged growth factor-induced heterodimerization. The B-Raf T753A mutant enhanced differentiation of PC12 cells, which was previously shown to be dependent on sustained ERK signaling. Fine mapping of the interaction sites by peptide arrays suggested a complex mode of interaction involving multiple contact sites with a main Raf-1 binding site in B-Raf encompassing T753. In summary, our data suggest that Raf-1/B-Raf heterodimerization occurs as part of the physiological activation process and that the heterodimer has distinct biochemical properties that may be important for the regulation of some biological processes.

The ablation of the protein kinase Raf-1 renders cells hypersensitive to apoptosis despite normal regulation of extracellular signal-regulated kinases, which suggests that apoptosis protection is mediated by a distinct pathway. We used proteomic analysis of Raf-1 signaling complexes to show that Raf-1 counteracts apoptosis by suppressing the activation of mammalian sterile 20-like kinase (MST2). Raf-1 prevents dimerization and phosphorylation of the activation loop of MST2 independently of its protein kinase activity. Depletion of MST2 from Raf-1-/- mouse or human cells abrogated sensitivity to apoptosis, whereas overexpression of MST2 induced apoptosis. Conversely, depletion of Raf-1 from Raf-1+/+ mouse or human cells led to MST2 activation and apoptosis. The concomitant depletion of both Raf-1 and MST2 prevented apoptosis.

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Ectopic expression of dual-specificity phosphatase 5 (DUSP5), an inducible mitogen-activated protein (MAP) kinase phosphatase, specifically inactivates and anchors extracellular signal-regulated kinase (ERK)1/2 in the nucleus. However, the role of endogenous DUSP5 in regulating the outcome of Ras/ERK kinase signaling under normal and pathological conditions is unknown. Here we report that mice lacking DUSP5 show a greatly increased sensitivity to mutant Harvey-Ras (HRas Q61L )-driven papilloma formation in the 7,12-Dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) model of skin carcinogenesis. Furthermore, mouse embryo fibroblasts (MEFs) from DUSP5 −/− mice show increased levels of nuclear phospho-ERK immediately after TPA stimulation and fail to accumulate total ERK in the nucleus compared with DUSP5 +/+ cells. Surprisingly, a microarray analysis reveals that only a small number of Ras/ERK-dependent TPA-responsive transcripts are up-regulated on deletion of DUSP5 in MEFs and mouse skin. The most up-regulated gene on DUSP5 loss encodes SerpinB2, an inhibitor of extracellular urokinase plasminogen activator and deletion of DUSP5 acts synergistically with mutant HRas Q61L and TPA to activate ERK-dependent SerpinB2 expression at the transcriptional level. SerpinB2 has previously been implicated as a mediator of DMBA/TPA-induced skin carcinogenesis. By analyzing DUSP5−/− double knockout mice, we demonstrate that deletion of SerpinB2 abrogates the increased sensitivity to papilloma formation seen on DUSP5 deletion. We conclude that DUSP5 performs a key nonredundant role in regulating nuclear ERK activation, localization, and gene expression. Furthermore, our results suggest an in vivo role for DUSP5 as a tumor suppressor by modulating the oncogenic potential of activated Ras in the epidermis.is one of four mammalian inducible, nuclear mitogen-activated protein kinase (MAPK) phosphatases (MKPs) (1). However, DUSP5 is unique within this group in targeting only the classical extracellular signal-regulated kinases 1 and 2 (referred to hereafter as ERK) (2). This, coupled with the finding that ERK activation is required for inducible DUSP5 expression, indicates that it acts as a negative feedback regulator of nuclear Ras/ERK signaling (3). DUSP5 overexpression also leads to nuclear accumulation of endogenous ERK (2), suggesting that DUSP5 may also act as a nuclear anchor, thus regulating both the spatial organization and activity of the pathway (4).Ras/ERK signaling is frequently deregulated in human cancers due to activating mutations in pathway components such as growth factor receptors, Ras GTPases, and the MAPK kinase kinase, BRaf (5). BRaf is mutated in 40-60% of malignant melanomas as well as in thyroid, colorectal, and lung tumors, underscoring the importance of this pathway and making it a focus of anticancer drug development (6). Whereas mechanisms of Ras/MAPK pathway activation in cancer are understood, little is known about how negative feedback controls influence tumorigenesis (7). Stud...

The authors wish to note the following: "Since the publication of our paper, we have become aware that the quantitative data presented in Fig. 1B do not represent the full raw data set provided in the supporting information deposited online at https://doi.org/ 10.15125/BATH-00317. We are therefore publishing a corrected version of Fig. 1 in which the graphs in panel B have been replaced by those generated from the complete online dataset. The result and the conclusions drawn are unchanged. A minor correction has also been made to the figure legend to reflect the altered P values that result from use of the complete dataset." The corrected Fig. 1 and its corrected legend appear below.

Inhibition of the androgen receptor (AR) is the main strategy to treat advanced prostate cancers. AR-independent treatmentresistant prostate cancer is a major unresolved clinical problem. Patients with prostate cancer with alterations in canonical WNT pathway genes, which lead to b-catenin activation, are refractory to AR-targeted therapies. Here, using clinically relevant murine prostate cancer models, we investigated the significance of b-catenin activation in prostate cancer progression and treatment resistance. b-Catenin activation, independent of the cell of origin, cooperated with Pten loss to drive AR-independent castrationresistant prostate cancer. Prostate tumors with b-catenin activation relied on the noncanonical WNT ligand WNT5a for sustained growth. WNT5a repressed AR expression and maintained the expression of c-Myc, an oncogenic effector of b-catenin activation, by mediating nuclear localization of NFkBp65 and b-catenin. Overall, WNT/b-catenin and AR signaling are reciprocally inhibited. Therefore, inhibiting WNT/b-catenin signaling by limiting WNT secretion in concert with AR inhibition may be useful for treating prostate cancers with alterations in WNT pathway genes. Significance: Targeting of both AR and WNT/b-catenin signaling may be required to treat prostate cancers that exhibit alterations of the WNT pathway.

Transplant outcomes for DCD and DBD kidneys are comparable. Baseline donor kidney disease influences DGF and graft survival but the impact is no greater for DCD kidneys.

BACKGROUND: Docetaxel chemotherapy in prostate cancer has a modest impact on survival. To date, efforts to develop combination therapies have not translated into new treatments. We sought to develop a novel therapeutic strategy to tackle chemoresistant prostate cancer by enhancing the efficacy of docetaxel. METHODS: We performed a drug-repurposing screen by using murine-derived prostate cancer cell lines driven by clinically relevant genotypes. Cells were treated with docetaxel alone, or in combination with drugs (n = 857) from repurposing libraries, with cytotoxicity quantified using High Content Imaging Analysis. RESULTS: Mebendazole (an anthelmintic drug that inhibits microtubule assembly) was selected as the lead drug and shown to potently synergise docetaxel-mediated cell killing in vitro and in vivo. Dual targeting of the microtubule structure was associated with increased G2/M mitotic block and enhanced cell death. Strikingly, following combined docetaxel and mebendazole treatment, no cells divided correctly, forming multipolar spindles that resulted in aneuploid daughter cells. Liposomes entrapping docetaxel and mebendazole suppressed in vivo prostate tumour growth and extended progression-free survival. CONCLUSIONS: Docetaxel and mebendazole target distinct aspects of the microtubule dynamics, leading to increased apoptosis and reduced tumour growth. Our data support a new concept of combined mebendazole/docetaxel treatment that warrants further clinical evaluation.