Metastasis underlies the majority of cancer-related deaths. Thus, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelialto-mesenchymal transitions (EMTs) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignant progression. Characterization of EMT effectors is likely to yield new insights into metastasis and novel avenues for treatment. We show that the presence of the receptor tyrosine kinase Axl in primary breast cancers independently predicts strongly reduced overall patient survival, and that matched patient metastatic lesions show enhanced Axl expression. We demonstrate that Axl is strongly induced by EMT in immortalized mammary epithelial cells that establishes an autocrine signaling loop with its ligand, Gas6. Epiallelic RNA interference analysis in metastatic breast cancer cells delineated a distinct threshold of Axl expression for mesenchymal-like in vitro cell invasiveness and formation of tumors in foreign and tissue-engineered microenvironments in vivo. Importantly, in two different optical imagingbased experimental breast cancer models, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells from the mammary gland to lymph nodes and several major organs and increased overall survival. These findings suggest that Axl represents a downstream effector of the tumor cell EMT that is required for breast cancer metastasis. Thus, the detection and targeted treatment of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer.carcinoma | receptor tyrosine kinase | breast cancer

The double-stranded RNA-binding domain (dsRBD) is a common RNA-binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The domain binds optimally to RNA stem-loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of bicoid and oskar mRNAs. Using high-resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem-loop. The dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix alpha1 interacts with the single-stranded loop that caps the RNA helix. Interactions between helix alpha1 and single-stranded RNA may be important determinants of the specificity of dsRBD proteins.

Carfilzomib is a proteasome inhibitor in clinical development that primarily targets the chymotrypsin-like (CT-L) subunits in both the constitutive proteasome (c20S) and the immunoproteasome (i20S). To investigate the impact of inhibiting the CT-L activity with carfilzomib, we set out to quantitate the levels of CT-L subunits ␤5 from the c20S and LMP7 from the i20S in normal and malignant hematopoietic cells. We found that the i20S is a major form of the proteasome expressed in cells of hematopoietic origin, including multiple myeloma (MM) CD138 ؉ tumor cells. Although specific inhibition of either LMP7 or ␤5 alone was insufficient to produce an antitumor response, inhibition of all proteasome subunits was cytotoxic to both hematologic tumor cells and peripheral blood mononuclear cells. However, selective inhibition of both ␤5 and LMP7 was sufficient to induce an antitumor effect in MM, non-Hodgkin lymphoma, and leukemia cells while minimizing the toxicity toward nontransformed cells. In MM tumor cells, CT-L inhibition alone was sufficient to induce proapoptotic sequelae, including proteasome substrate accumulation, Noxa and caspase 3/7 induction, and phospho-eIF2␣ suppression. These data support a hypothesis that hematologic tumor cells are uniquely sensitive to CT-L inhibition and provide a mechanistic understanding of the clinical safety profile and antitumor activity of proteasome inhibitors. (Blood.

Neuroblasts undergo asymmetric stem cell divisions to generate a series of ganglion mother cells (GMCs). During these divisions, the cell fate determinant Prospero is asymmetrically partitioned to the GMC by Miranda protein, which tethers it to the basal cortex of the dividing neuroblast. Interestingly, prospero

The mago nashi gene plays two essential roles in Drosophila axis formation: it is required downstream of the signal from the posterior follicle cells for the polarisation of the oocyte microtubule cytoskeleton, and has a second, independent role in the localisation of oskar mRNA to the posterior of the oocyte.

Drosophila Staufen protein is required for the localization of oskar mRNA to the posterior of the oocyte, the anterior anchoring of bicoid mRNA and the basal localization of prospero mRNA in dividing neuroblasts. The only regions of Staufen that have been conserved throughout animal evolution are five double-stranded (ds)RNA-binding domains (dsRBDs) and a short region within an insertion that splits dsRBD2 into two halves. dsRBDs 1, 3 and 4 bind dsRNA in vitro, but dsRBDs 2 and 5 do not, although dsRBD2 does bind dsRNA when the insertion is removed. Full-length Staufen protein lacking this insertion is able to associate with oskar mRNA and activate its translation, but fails to localize the RNA to the posterior. In contrast, Staufen lacking dsRBD5 localizes oskar mRNA normally, but does not activate its translation. Thus, dsRBD2 is required for the microtubule-dependent localization of osk mRNA, and dsRBD5 for the derepression of oskar mRNA translation, once localized. Since dsRBD5 has been shown to direct the actin-dependent localization of prospero mRNA, distinct domains of Staufen mediate microtubule- and actin-based mRNA transport.

BackgroundBlood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells) and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics.Methods and FindingsTo better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF.ConclusionsThese results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation.

Repurposing ‘old’ drugs can facilitate rapid clinical translation but necessitates novel mechanistic insight. Warfarin, a vitamin K “antagonist” used clinically for the prevention of thrombosis for over 50 years, has been shown to have anti-cancer effects. We hypothesized that the molecular mechanism underlying its anti-tumor activity is unrelated to its effect on coagulation, but is due to inhibition of the Axl receptor tyrosine kinase on tumor cells. Activation of Axl by its ligand Gas6, a vitamin K-dependent protein, is inhibited at doses of warfarin that do not affect coagulation. Here we show that inhibiting Gas6-dependent Axl activation with low dose warfarin or with other tumor-specific Axl targeting agents, blocks the progression and spread of pancreatic cancer. Warfarin also inhibited Axl-dependent tumor cell migration, invasiveness and proliferation while increasing apoptosis and sensitivity to chemotherapy. We conclude that Gas6-induced Axl signaling is a critical driver of pancreatic cancer progression and its inhibition with low dose warfarin or other Axl targeting agents may improve outcome in patients with Axl-expressing tumors.