CaMKK2 facilitates Golgi-associated vesicle trafficking to sustain cancer cell proliferation.

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) regulates cell and whole-body metabolism and supports tumorigenesis. The cellular impacts of perturbing CAMKK2 expression are, however, not yet fully characterised. By knocking down CAMKK2 levels, we have identified a number of significant subcellular changes indicative of perturbations in vesicle trafficking within the endomembrane compartment. To determine how they might contribute to effects on cell proliferation, we have used proteomics to identify Gemin4 as a direct interactor, capable of binding CAMKK2 and COPI subunits. Prompted by this, we confirmed that CAMKK2 knockdown leads to concomitant and significant reductions in δ-COP protein. Using imaging, we show that CAMKK2 knockdown leads to Golgi expansion, the induction of ER stress, abortive autophagy and impaired lysosomal acidification. All are phenotypes of COPI depletion. Based on our findings, we hypothesise that CAMKK2 sustains cell proliferation in large part through effects on organelle integrity and membrane trafficking.

Molecular basis of USP7 inhibition by selective small-molecule inhibitors.

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.

Soluble Robo4 receptor inhibits in vivo angiogenesis and endothelial cell migration.

Roundabout receptors are molecular guidance molecules that function by interaction with Slit proteins to regulate axon guidance, neuronal migration, and leukocyte chemotaxis. We recently isolated a novel roundabout gene, called Robo4, which is restricted in expression to the endothelium, notably in areas of angiogenesis. The aim of this study was to use the soluble extracellular domain of Robo4 as a probe of function in angiogenesis and endothelial biology. Thus, the soluble extracellular domain of the receptor (Robo4Fc) showed diverse in vivo and in vitro activities including 1) inhibition of angiogenesis in vivo in the rodent subcutaneous sponge model, 2) inhibition of tube formation in the rat aortic ring assay, 3) inhibition of VEGF- and bFGF-stimulated endothelial cell migration, and 4) inhibition of endothelial proliferation. To assess whether Robo4Fc was inhibiting Slit-mediated effects, we determined whether Robo4 and Slit interact. Recombinant Slits-1, -2, and -3 were shown by immunoprecipitation and BiaCore analysis to bind to Robo1 but not Robo4. Further study of the role of Robo4 in angiogenesis appears justified.