A novel, safe, fast and efficient treatment for Her2-positive and negative bladder cancer utilizing an EGF-anthrax toxin chimera.

Bladder cancer is the sixth most common cancer in the United States, and it exhibits an alarming 70% recurrence rate. Thus, the development of more efficient antibladder cancer approaches is a high priority. Accordingly, this work provides the basis for a transformative anticancer strategy that takes advantage of the unique characteristics of the bladder. Unlike mucin-shielded normal bladder cells, cancer cells are exposed to the bladder lumen and overexpress EGFR. Therefore, we used an EGF-conjugated anthrax toxin that after targeting EGFR was internalized and triggered apoptosis in exposed bladder cancer cells. This unique agent presented advantages over other EGF-based technologies and other toxin-derivatives. In contrast to known agents, this EGF-toxin conjugate promoted its own uptake via receptor microclustering even in the presence of Her2 and induced cell death with a LC50 < 1 nM. Furthermore, our data showed that exposures as short as ≈3 min were enough to commit human (T24), mouse (MB49) and canine (primary) bladder cancer cells to apoptosis. Exposure of tumor-free mice and dogs with the agent resulted in no toxicity. In addition, the EGF-toxin was able to eliminate cells from human patient tumor samples. Importantly, the administration of EGF-toxin to dogs with spontaneous bladder cancer, who had failed or were not eligible for other therapies, resulted in ~30% average tumor reduction after one treatment cycle. Because of its in vitro and in vivo high efficiency, fast action (reducing treatment time from hours to minutes) and safety, we propose that this EGF-anthrax toxin conjugate provides the basis for new, transformative approaches against bladder cancer.

Granulocyte colony-stimulating factor (G-CSF) upregulates ß1 integrin and increases migration of human trophoblast Swan 71 cells via PI3K and MAPK activation.

Multiple cytokines and growth factors expressed at the fetal-maternal interface are involved in the regulation of trophoblast functions and placental growth, but the role of G-CSF has not been completely established. Based on our previous study showing that G-CSF increases the activity of matrix metalloproteinase-2 and the release of vascular endothelial growth factor in Swan 71 human trophoblast cells, in this work we explore the possible contribution of G-CSF to cell migration and the G-CSF-triggered signaling pathway. We found that G-CSF induced morphological changes on actin cytoskeleton consistent with a migratory cell phenotype. G-CSF also up-regulated the expression levels of ß1 integrin and promoted Swan 71 cell migration. By using selective pharmacological inhibitors and dominant negative mutants we showed that PI3K, Erk 1/2 and p38 pathways are required for promoting Swan 71 cell motility. It was also demonstrated that PI3K behaved as an upstream regulator of Erk 1/2 and p38 MAPK. In addition, the increase of ß1 integrin expression was dependent on PI3K activation. In conclusion, our results indicate that G-CSF stimulates ß1 integrin expression and Swan 71 cell migration by activating PI3K and MAPK signaling pathways, suggesting that G-CSF should be considered as an additional regulatory factor that contributes to a successful embryo implantation and to the placenta development.

Epsin N-terminal homology domains perform an essential function regulating Cdc42 through binding Cdc42 GTPase-activating proteins.

Epsins are endocytic proteins with a structured epsin N-terminal homology (ENTH) domain that binds phosphoinositides and a poorly structured C-terminal region that interacts with ubiquitin and endocytic machinery, including clathrin and endocytic scaffolding proteins. Yeast has two redundant genes encoding epsins, ENT1 and ENT2; deleting both genes is lethal. We demonstrate that the ENTH domain is both necessary and sufficient for viability of ent1Deltaent2Delta cells. Mutational analysis of the ENTH domain revealed a surface patch that is essential for viability and that binds guanine nucleotide triphosphatase-activating proteins for Cdc42, a critical regulator of cell polarity in all eukaryotes. Furthermore, the epsins contribute to regulation of specific Cdc42 signaling pathways in yeast cells. These data support a model in which the epsins function as spatial and temporal coordinators of endocytosis and cell polarity.

Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor.

The cation-independent mannose 6-phosphate receptor (CI-MPR) mediates sorting of lysosomal hydrolase precursors from the TGN to endosomes. After releasing the hydrolase precursors into the endosomal lumen, the unoccupied receptor returns to the TGN for further rounds of sorting. Here, we show that the mammalian retromer complex participates in this retrieval pathway. The hVps35 subunit of retromer interacts with the cytosolic domain of the CI-MPR. This interaction probably occurs in an endosomal compartment, where most of the retromer is localized. In particular, retromer is associated with tubular-vesicular profiles that emanate from early endosomes or from intermediates in the maturation from early to late endosomes. Depletion of retromer by RNA interference increases the lysosomal turnover of the CI-MPR, decreases cellular levels of lysosomal hydrolases, and causes swelling of lysosomes. These observations indicate that retromer prevents the delivery of the CI-MPR to lysosomes, probably by sequestration into endosome-derived tubules from where the receptor returns to the TGN.