The Role of BCA2 in the Endocytic Trafficking of EGFR and Significance as a Prognostic Biomarker in Cancer.

Breast Cancer Associated gene 2 (BCA2) is an E3 ubiquitin ligase that is over-expressed in >50% of primary breast cancers, and has been shown to increase in vitro cell proliferation and invasion. The protein has been linked to alterations in EGFR degradation; however there is some dispute as to its role and influence on the biology of this receptor. Our work aimed to ascertain the role of BCA2 in EGFR endocytosis and down-regulation and to examine its links with breast cancer outcome. Data generated with the online expression analysis tool KM-Plotter showed that high BCA2 levels are associated with poor prognosis in ovarian, gastric and breast cancer, particularly HER2 over-expressing breast cancers. Experimentally, we demonstrate that over-expression of BCA2 induced a reduction in total EGFR levels. BCA2 over-expressing cells stimulated with EGF exhibited reduced lysosomal degradation of both this ligand and its receptor. Signalling downstream of EGFR in BCA2 over-expressing cells was characterized by a lower magnitude but increased duration. Our findings support a role for BCA2 in receptor endocytosis. Consistent with this we show that BCA2 over-expression reduces the level of vesicle-associated Rab7, a regulator of late endocytosis and documented interaction partner of BCA2. Levels of transferrin receptor and the uptake of transferrin were unaltered by over-expression of BCA2 indicating that trafficking changes may be limited to late endocytic sorting events. This report offers a thorough exploration of BCA2 biology and suggests a context-dependent role for the protein in the endocytic regulation of EGFR and as a prognostic biomarker in cancer.

Simple and Versatile 3D Printed Microfluidics Using Fused Filament Fabrication.

The uptake of microfluidics by the wider scientific community has been limited by the fabrication barrier created by the skills and equipment required for the production of traditional microfluidic devices. Here we present simple 3D printed microfluidic devices using an inexpensive and readily accessible printer with commercially available printer materials. We demonstrate that previously reported limitations of transparency and fidelity have been overcome, whilst devices capable of operating at pressures in excess of 2000 kPa illustrate that leakage issues have also been resolved. The utility of the 3D printed microfluidic devices is illustrated by encapsulating dental pulp stem cells within alginate droplets; cell viability assays show the vast majority of cells remain live, and device transparency is sufficient for single cell imaging. The accessibility of these devices is further enhanced through fabrication of integrated ports and by the introduction of a Lego®-like modular system facilitating rapid prototyping whilst offering the potential for novices to build microfluidic systems from a database of microfluidic components.

Novel cis-selective and non-epimerisable C3 hydroxy azapodophyllotoxins targeting microtubules in cancer cells.

Podophyllotoxin (PT) and its clinically used analogues are known to be powerful antitumour agents. These compounds contain a trans fused strained γ-lactone system, a feature that correlates to the process of epimerisation, whereby the trans γ-lactone system of ring D opens and converts to the more thermodynamically stable cis epimer. Since these cis epimers are known to be either less active or lacking antitumour activity, epimerisation is an undesirable feature from a chemotherapeutic point of view. To circumvent this problem, considerable efforts have been reported, amongst which is the synthesis of azapodophyllotoxins where the stereocentres at C2 and C3 are removed in order to preclude epimerisation. Herein we report the identification of a novel C3 hydroxy, cis-selective γ-lactone configuration of ring C in the azapodophyllotoxin scaffold, through an efficient stereoselective multicomponent reaction (MCR) involving fluorinated and non-fluorinated aldehydes. This configuration releases the highly strained trans γ-lactone system in podophyllotoxin analogues into the more thermodynamically stable cis γ-lactone motif and yet retains significantly potent activity. These compounds were evaluated against the human cancer lines MCF-7 and 22Rv1 in vitro. Fourteen out of the seventeen tested compounds exhibited sub-micromolar activity with IC50 values in the range of 0.11-0.91 µM, which is comparable and in some cases better than the activity profile of etoposide in this assay. Interestingly, we obtained strong evidence from spectroscopic and X-ray data analyses that the previously reported structure of similar analogues is not accurate. Molecular modelling performed using the podophyllotoxin binding site on ß tubulin revealed a novel binding mode of these analogues. Furthermore, sub-cellular study of our compounds using immunolabelling and confocal microscopy analyses showed strong microtubule disruptive activity, particularly in dividing cells.

siRNA Inhibition of Endocytic Pathways to Characterize the Cellular Uptake Mechanisms of Folate-Functionalized Glycol Chitosan Nanogels.

Glycol chitosan nanogels have been widely used in gene, drug, and contrast agent delivery in an effort to improve disease diagnosis and treatment. Herein, we evaluate the internalization mechanisms and intracellular fate of previously described glycol chitosan nanogels decorated with folate to target the folate receptor. Uptake of the folate-decorated nanogel was impaired by free folate, suggesting competitive inhibition and shared internalization mechanisms via the folate receptor. Nanogel uptake was shown to occur mainly through flotillin-1 and Cdc42-dependent endocytosis. This was determined by inhibition of uptake reduction observed upon siRNA depletion of these two proteins and the pathways that they regulate. The data also suggest the involvement of the actin cytoskeleton in nanogel uptake via macropinocytosis. After 7 h of incubation with HeLa cells, approximately half of the nanogel population was localized in endolysosomal compartments, whereas the remaining 50% of the material was in undefined regions of the cytoplasm. Glycol chitosan nanogels may thus have potential as drug delivery vectors for targeting different intracellular compartments.

Disulfiram-induced cytotoxicity and endo-lysosomal sequestration of zinc in breast cancer cells.

Disulfiram, a clinically used alcohol-deterrent has gained prominence as a potential anti-cancer agent due to its impact on copper-dependent processes. Few studies have investigated zinc effects on disulfiram action, despite it having high affinity for this metal. Here we studied the cytotoxic effects of disulfiram in breast cancer cells, and its relationship with both intra and extracellular zinc. MCF-7 and BT474 cancer cell lines gave a striking time-dependent biphasic cytotoxic response between 0.01 and 10 µM disulfiram. Co-incubation of disulfiram with low-level zinc removed this effect, suggesting that availability of extracellular zinc significantly influences disulfiram efficacy. Live-cell confocal microscopy using fluorescent endocytic probes and the zinc dye Fluozin-3 revealed that disulfiram selectively and rapidly increased zinc levels in endo-lysosomes. Disulfiram also caused spatial disorganization of late endosomes and lysosomes, suggesting they are novel targets for this drug. This relationship between disulfiram toxicity and ionophore activity was consolidated via synthesis of a new disulfiram analog and overall we demonstrate a novel mechanism of disulfiram-cytotoxicity with significant clinical implications for future use as a cancer therapeutic.