The molecular and clinical role of Tensin 1/2/3 in cancer.

Tensin 1 was originally described as a focal adhesion adaptor protein, playing a role in extracellular matrix and cytoskeletal interactions. Three other Tensin proteins were subsequently discovered, and the family was grouped as Tensin. It is now recognized that these proteins interact with multiple cell signalling cascades that are implicated in tumorigenesis. To understand the role of Tensin 1-3 in neoplasia, current molecular evidence is categorized by the hallmarks of cancer model. Additionally, clinical data involving Tensin 1-3 are reviewed to investigate the correlation between cellular effects and clinical phenotype. Tensin proteins commonly interact with the tumour suppressor, DLC1. The ability of Tensin to promote tumour progression is directly correlated with DLC1 expression. Members of the Tensin family appear to have tumour subtype-dependent effects on oncogenesis; despite numerous data evidencing a tumour suppressor role for Tensin 2, association of Tensins 1-3 with an oncogenic role notably in colorectal carcinoma and pancreatic ductal adenocarcinoma is of potential clinical relevance. The complex interplay between these focal adhesion adaptor proteins and signalling pathways are discussed to provide an up to date review of their role in cancer biology.

Transient regulation of focal adhesion via Tensin3 is required for nascent oligodendrocyte differentiation.

The differentiation of oligodendroglia from oligodendrocyte precursor cells (OPCs) to complex and extensive myelinating oligodendrocytes (OLs) is a multistep process that involves large-scale morphological changes with significant strain on the cytoskeleton. While key chromatin and transcriptional regulators of differentiation have been identified, their target genes responsible for the morphological changes occurring during OL myelination are still largely unknown. Here, we show that the regulator of focal adhesion, Tensin3 (Tns3), is a direct target gene of Olig2, Chd7, and Chd8, transcriptional regulators of OL differentiation. Tns3 is transiently upregulated and localized to cell processes of immature OLs, together with integrin-ß1, a key mediator of survival at this transient stage. Constitutive <i>Tns3</i> loss of function leads to reduced viability in mouse and humans, with surviving knockout mice still expressing Tns3 in oligodendroglia. Acute deletion of <i>Tns3</i> in vivo, either in postnatal neural stem cells (NSCs) or in OPCs, leads to a twofold reduction in OL numbers. We find that the transient upregulation of Tns3 is required to protect differentiating OPCs and immature OLs from cell death by preventing the upregulation of p53, a key regulator of apoptosis. Altogether, our findings reveal a specific time window during which transcriptional upregulation of Tns3 in immature OLs is required for OL differentiation likely by mediating integrin-ß1 survival signaling to the actin cytoskeleton as OL undergo the large morphological changes required for their terminal differentiation.

The first laminin G-like domain of protein S is essential for binding and activation of Tyro3 receptor and intracellular signalling.

The homologous proteins Gas6 and protein S (ProS1) are both natural ligands for the TAM (Tyro3, Axl, MerTK) receptor tyrosine kinases. ProS1 selectively activates Tyro3; however, the precise molecular interface of the ProS1-Tyro3 contact has not been characterised. We used a set of chimeric proteins in which each of the C-terminal laminin G-like (LG) domains of ProS1 were swapped with those of Gas6, as well as a set of ProS1 mutants with novel added glycosylations within LG1. Alongside wildtype ProS1, only the chimera containing ProS1 LG1 domain stimulated Tyro3 and Erk phosphorylation in human cancer cells, as determined by Western blot. In contrast, Gas6 and chimeras containing minimally the Gas6 LG1 domain stimulated Axl and Akt phosphorylation. We performed in silico homology modelling and molecular docking analysis to construct and evaluate structural models of both ProS1-Tyro3 and Gas6-Axl ligand-receptor interactions. These analyses revealed a contact between the ProS1 LG1 domain and the first immunoglobulin domain of Tyro3, which was similar to the Gas6-Axl interaction, and involved long-range electrostatic interactions that were further stabilised by hydrophobic and polar contacts. The mutant ProS1 proteins, which had added glycosylations within LG1 but which were all outside of the modelled contact region, all activated Tyro3 in cells with no hindrance. In conclusion, we show that the LG1 domain of ProS1 is necessary for activation of the Tyro3 receptor, involving protein-protein interaction interfaces that are homologous to those of the Gas6-Axl interaction.

Gas6/TAM Signalling Negatively Regulates Inflammatory Induction of GM-CSF in Mouse Brain Microglia.

Microglia and astrocytes are the main CNS glial cells responsible for the neuroinflammatory response, where they release a plethora of cytokines into the CNS inflammatory milieu. The TAM (Tyro3, Axl, Mer) receptors and their main ligand Gas6 are regulators of this response, however, the underlying mechanisms remain to be determined. We investigated the ability of Gas6 to modulate the CNS glial inflammatory response to lipopolysaccharide (LPS), a strong pro-inflammatory agent, through a qPCR array that explored Toll-like receptor signalling pathway-associated genes in primary cultured mouse microglia. We identified the Csf2 gene, encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), as a major Gas6 target gene whose induction by LPS was markedly blunted by Gas6. Both the Csf2 gene induction and the suppressive effect of Gas6 on this were emulated through measurement of GM-CSF protein release by cells. We found distinct profiles of GM-CSF induction in different glial cell types, with microglia being most responsive during inflammation. Also, Gas6 markedly inhibited the LPS-stimulated nuclear translocation of NF-κB p65 protein in microglia. These results illustrate microglia as a major resident CNS cellular source of GM-CSF as part of the neuroinflammatory response, and that Gas6/TAM signalling inhibits this response through suppression of NF-κB signalling.

Inhibition of DCLK1 with DCLK1-IN-1 Suppresses Renal Cell Carcinoma Invasion and Stemness and Promotes Cytotoxic T-Cell-Mediated Anti-Tumor Immunity.

The approval of immune checkpoint inhibitors has expanded treatment options for renal cell carcinoma (RCC), but new therapies that target RCC stemness and promote anti-tumor immunity are needed. Previous findings demonstrate that doublecortin-like kinase 1 (DCLK1) regulates stemness and is associated with RCC disease progression. Herein, we demonstrate that small-molecule kinase inhibitor DCLK1-IN-1 strongly inhibits DCLK1 phosphorylation and downregulates pluripotency factors and cancer stem cell (CSC) or epithelial-mesenchymal transition (EMT)-associated markers including c-MET, c-MYC, and N-Cadherin in RCC cell lines. Functionally, DCLK1-IN-1 treatment resulted in significantly reduced colony formation, migration, and invasion. Additionally, assays using floating or Matrigel spheroid protocols demonstrated potent inhibition of stemness. An analysis of clinical populations showed that DCLK1 predicts RCC survival and that its expression is correlated with reduced CD8+ cytotoxic T-cell infiltration and increases in M2 immunosuppressive macrophage populations. The treatment of RCC cells with DCLK1-IN-1 significantly reduced the expression of immune checkpoint ligand PD-L1, and co-culture assays using peripheral blood monocytes (PBMCs) or T-cell expanded PBMCs demonstrated a significant increase in immune-mediated cytotoxicity alone or in combination with anti-PD1 therapy. Together, these findings demonstrate broad susceptibility to DCLK1 kinase inhibition in RCC using DCLK1-IN-1 and provide the first direct evidence for DCLK1-IN-1 as an immuno-oncology agent.

Functional succinate dehydrogenase deficiency is a common adverse feature of clear cell renal cancer.

Reduced succinate dehydrogenase (SDH) activity resulting in adverse succinate accumulation was previously considered relevant only in 0.05 to 0.5% of kidney cancers associated with germline SDH mutations. Here, we sought to examine a broader role for SDH loss in kidney cancer pathogenesis/progression. We report that underexpression of SDH subunits resulting in accumulation of oncogenic succinate is a common feature in clear cell renal cell carcinoma (ccRCC) (∼80% of all kidney cancers), with a marked adverse impact on survival in ccRCC patients (n = 516). We show that SDH down-regulation is a critical brake in the TCA cycle during ccRCC pathogenesis and progression. In exploring mechanisms of SDH down-regulation in ccRCC, we report that Von Hippel-Lindau loss-induced hypoxia-inducible factor-dependent up-regulation of miR-210 causes direct inhibition of the SDHD transcript. Moreover, shallow deletion of SDHB occurs in ∼20% of ccRCC. We then demonstrate that SDH loss-induced succinate accumulation contributes to adverse loss of 5-hydroxymethylcytosine, gain of 5-methylcytosine, and enhanced invasiveness in ccRCC via inhibition of ten-eleven translocation (TET)-2 activity. Intriguingly, binding affinity between the catalytic domain of recombinant TET-2 and succinate was found to be very low, suggesting that the mechanism of succinate-induced attenuation of TET-2 activity is likely via product inhibition rather than competitive inhibition. Finally, exogenous ascorbic acid, a TET-activating demethylating agent, led to reversal of the above oncogenic effects of succinate in ccRCC cells. Collectively, our study demonstrates that functional SDH deficiency is a common adverse feature of ccRCC and not just limited to the kidney cancers associated with germline SDH mutations.

Identification of signalling pathways activated by Tyro3 that promote cell survival, proliferation and invasiveness in human cancer cells.

Tyro3 is a member of the TAM subfamily of receptor tyrosine kinases alongside Axl and MerTK, which are activated by homologous ligands Gas6 and protein S. The TAMs activate signalling pathways that mediate diverse functions including cell survival, proliferation, phagocytosis and immune regulation, and defects in TAM-dependent processes are associated with the development of human autoimmune diseases and numerous cancers. In this study, we have focused on the signalling and functional roles of Tyro3, about which much remains unknown. For this purpose, we used cultured human cancer cell lines with different levels of TAM expression to reveal the relative significance of Tyro3 amongst the TAMs. Knockdown of Tyro3 expression by siRNA in MGH-U3 cells, which express Tyro3 as sole TAM, caused a reduction in cell viability, which could not be rescued by TAM ligand, demonstrating the dependence of these cells solely on Tyro3. In contrast, the reduced viability of SCC-25 cells upon Tyro3 knockdown could be rescued by Gas6 as these cells express both Tyro3 and Axl and hence Axl expression was preserved. An increase in the fraction of Tyro3 knockdown cells in the early apoptotic phase was observed in four different cell lines each with a different TAM expression profile, revealing a broad anti-apoptotic function of Tyro3. Furthermore, in the Tyro3-dependent cells, Tyro3 depletion caused a significant increase in cells in the G0/G1 phase of the cell cycle concomitant with decreases in the G2/M and S phases. In addition, a cancer pathway gene discovery array revealed distinct sets of genes that were altered in expression in cancer cells upon Tyro3 knockdown. Together, these results have elucidated further a role of Tyro3 in promoting multiple tumour-supporting pathways in human cancer cells, which differs in extent depending on the presence of other TAMs in the same cells.

Immune evasion in renal cell carcinoma: biology, clinical translation, future directions.

Targeted therapies and immune checkpoint inhibitors have advanced the treatment landscape of Renal Cell Carcinoma (RCC) over the last decade. While checkpoint inhibitors have demonstrated survival benefit and are currently approved in the front-line and second-line settings, primary and secondary resistance is common. A comprehensive understanding of the mechanisms of immune evasion in RCC is therefore critical to the development of effective combination treatment strategies. This article reviews the current understanding of the different, yet coordinated, mechanisms adopted by RCC cells to evade immune killing; summarizes various aspects of clinical translation thus far, including the currently registered RCC clinical trials exploring agents in combination with checkpoint inhibitors; and provides perspectives on the current landscape and future directions for the field.

Gas6 Inhibits Toll-Like Receptor-Mediated Inflammatory Pathways in Mouse Microglia via Axl and Mer.

Background: Microglia are well known key regulators of neuroinflammation which feature in multiple neurodegenerative disorders. These cells survey the CNS and, under inflammatory conditions, become "activated" through stimulation of toll-like receptors (TLRs), resulting in changes in morphology and production and release of cytokines. In the present study, we examined the roles of the related TAM receptors, Mer and Axl, and of their ligand, Gas6, in the regulation of microglial pro-inflammatory TNF-α production and microglial morphology. Methods: Primary cultures of murine microglia of wild-type (WT), Mer-/- and Axl-/- backgrounds were stimulated by the TLR4 agonist, lipopolysaccharide (LPS) with or without pre-treatment with Gas6. Gene expression of TNF-α, Mer, and Axl was examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) was used to measure TNF-α release from microglia. Immunofluorescence staining of ß-actin and the microglial marker Iba1 was performed to reveal microglial morphological changes, with cellular characteristics (area, perimeter, Feret's diameter, minimum Feret, roundness, and aspect ratio) being quantified using ImageJ software. Results: Under basal conditions, TNF-α gene expression was significantly lower in Axl-/- microglia compared to WT cells. However, all microglial cultures robustly responded to LPS stimulation with the upregulation of TNF-α expression to similar degrees. Furthermore, Mer receptor expression was less responsive to LPS stimulation when in Axl knockout cells. The presence of Gas6 consistently inhibited the LPS-induced upregulation of TNF-α in WT, Mer-/- and Axl-/- microglia. Moreover, Gas6 also inhibited LPS-induced changes in the microglial area, perimeter length, and cell roundness in wild-type cells. Conclusion: Gas6 can negatively regulate the microglial pro-inflammatory response to LPS as well as via stimulation of other TLRs, acting through either of the TAM receptors, Axl and Mer. This finding indicates an interaction between TLR and TAM receptor signaling pathways and reveals an anti-inflammatory role for the TAM ligand, Gas6, which could have therapeutic potential.

CTEN Induces Tumour Cell Invasion and Survival and Is Prognostic in Radiotherapy-Treated Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) is a heterogenous disease treated with surgery and/or (chemo) radiotherapy, but up to 50% of patients with late-stage disease develop locoregional recurrence. Determining the mechanisms underpinning treatment resistance could identify new therapeutic targets and aid treatment selection. C-terminal tensin-like (CTEN) is a member of the tensin family, upregulated in several cancers, although its expression and function in HNSCC are unknown. We found that CTEN is commonly upregulated in HNSCC, particularly HPV-ve tumours. In vitro CTEN was upregulated in HPV-ve (n = 5) and HPV+ve (n = 2) HNSCC cell lines. Stable shRNA knockdown of CTEN in vivo significantly reduced tumour growth (SCC-25), and functional analyses in vitro showed that CTEN promoted tumour cell invasion, colony formation and growth in 3D-culture (SCC-25, Detroit 562). RNA sequencing of SCC-25 cells following CTEN siRNA knockdown identified 349 differentially expressed genes (logFC > 1, p < 0.05). Gene ontology analysis highlighted terms relating to cell locomotion and apoptosis, consistent with in vitro findings. A membrane-based antibody array confirmed that CTEN regulated multiple apoptosis-associated proteins, including HSP60 and cleaved caspase-3. Notably, in a mixed cohort of HPV+ve and HPV-ve HNSCC patients (n = 259), we found a significant, independent negative association of CTEN with prognosis, limited to those patients treated with (chemo)radiotherapy, not surgery, irrespective of human papillomavirus (HPV) status. These data show that CTEN is commonly upregulated in HNSCC and exerts several functional effects. Its potential role in modulating apoptotic response to therapy suggests utility as a predictive biomarker or radio-sensitising target.

Gas6 Induces Myelination through Anti-Inflammatory IL-10 and TGF-ß Upregulation in White Matter and Glia.

The Gas6-TAM (Tyro3, Axl, Mer) ligand-receptor system is believed to promote central nervous system (CNS) (re)myelination and glial cell development. An additional important function of Gas6-TAM signalling appears to be the regulation of immunity and inflammation, which remains to be fully elucidated in the CNS. Here, we characterised the expression of TAM receptors and ligands in individual CNS glial cell types, observing high expression of Gas6 and the TAM receptors, Mer and Axl, in microglia, and high expression of Tyro3 in astrocytes. We also investigated the effect of Gas6 on the inflammatory cytokine response in the optic nerve and in mixed glial cell cultures from wildtype and single TAM receptor knockout mice. In wildtype and Mer-deficient cultures, Gas6 significantly stimulated the expression of the anti-inflammatory/pro-repair cytokines interleukin 10 (IL-10) and transforming growth factor ß (TGF-ß), whereas this effect was absent in either Tyro3 or Axl knockout cultures. Furthermore, Gas6 caused upregulation of myelin basic protein (MBP) expression in optic nerves, which was blocked by a neutralising antibody against IL-10. In conclusion, our data show that microglia are both a major source of Gas6 as well as an effector of Gas6 action in the CNS through the upregulation of anti-inflammatory and pro-repair mediators. Furthermore, the presence of both Axl and Tyro3 receptors appears to be necessary for these effects of Gas6. In addition, IL-10, alongside suppressing inflammation and immunity, mediates the pro-myelinating mechanism of Gas6 action in the optic nerve. Therefore, Gas6 may present an attractive target for novel therapeutic interventions for demyelinating as well as neuroinflammatory disorders of the CNS.

Galectin-3 Stimulates Tyro3 Receptor Tyrosine Kinase and Erk Signalling, Cell Survival and Migration in Human Cancer Cells.

The TAM (Tyro3, Axl, MerTK) subfamily of receptor tyrosine kinases (RTKs) and their ligands, Gas6 and protein S (ProS1), are implicated in tumorigenesis and chemoresistance in various cancers. The ß-galactoside binding protein galectin-3 (Gal-3), which is also implicated in oncogenesis, has previously been shown to be a ligand for MerTK. However, the selectivity of Gal-3 for the other TAM receptors, and its TAM-mediated signalling and functional properties in cancer cells, remain to be explored. The present study was aimed at determining these, including through direct comparison of Gal-3 with the two canonical TAM ligands. Exogenous Gal-3 rapidly stimulated Tyro3 receptor phosphorylation to the same extent as the Tyro3 ligand ProS1, but not Axl, in the cultured human cancer cell lines SCC-25 (express both Tyro3 and Axl) and MGH-U3 (express Tyro3 only). Gal-3 also activated intracellular Erk and Akt kinases in both cell lines and furthermore protected cells from acute apoptosis induced by staurosporine but not from serum-starvation induced apoptosis. In addition, Gal-3 significantly stimulated cancer cell migration rate in the presence of the Axl blocker BGB324. Therefore, these results have shown Gal-3 to be a novel agonist for Tyro3 RTK, activating a Tyro3-Erk signalling axis, as well as Akt signalling, in cancer cells that promotes cell survival, cell cycle progression and cell migration. These data therefore reveal a novel mechanism of Tyro3 RTK activation through the action of Gal-3 that contrasts with those of the known TAM ligands Gas6 and ProS1.

Tumour-Secreted Protein S (ProS1) Activates a Tyro3-Erk Signalling Axis and Protects Cancer Cells from Apoptosis.

The TAM subfamily (Tyro3, Axl, MerTK) of receptor tyrosine kinases are implicated in several cancers, where they have been shown to support primary tumorigenesis as well as secondary resistance to cancer therapies. Relatively little is known about the oncogenic role of Tyro3, including its ligand selectivity and signalling in cancer cells. Tyro3 showed widespread protein and mRNA expression in a variety of human cancer cell lines. In SCC-25 head and neck cancer cells expressing both Tyro3 and Axl, Western blotting showed that both natural TAM ligands ProS1 and Gas6 rapidly stimulated Tyro3 and Erk kinase phosphorylation, with ProS1 eliciting a greater effect. In contrast, Gas6 was the sole stimulator of Axl and Akt kinase phosphorylation. In MGH-U3 bladder cancer cells, which express Tyro3 alone, ProS1 was again the stronger stimulator of Tyro3 and Erk stimulation but additionally stimulated Akt phosphorylation. Conditioned medium from ProS1-secreting 786-0 kidney cancer cells replicated the kinase activation effects of recombinant ProS1 in SCC-25 cells, with specificity confirmed by ProS1 ligand traps and warfarin. In addition, ProS1 protected cancer cells from acute apoptosis induced by staurosporine, as well as additionally, long-term serum starvation-induced apoptosis in MGH-U3 cells (Tyro3 only), which reflects its additional coupling to Akt signalling in these cells. In conclusion, we have shown that ProS1 is a tumour-derived functional ligand for Tyro3 that supports cancer cell survival. Furthermore, the ProS1-Tyro3 interaction is primarily coupled to Erk signalling although it displays signalling diversity dependent upon its representative expression as a TAM receptor in tumour cells.

TAM Receptor Tyrosine Kinases in Cancer Drug Resistance.

Receptor tyrosine kinases (RTK) are major regulators of key biological processes, including cell growth, survival, and differentiation, and were established early on as proto-oncogenes, with aberrant expression linked to tumor progression in many cancers. Therefore, RTKs have emerged as major targets for selective therapy with small-molecule inhibitors. However, despite improvements in survival rates, it is now apparent that the targeting of RTKs with selective inhibitors is only transiently effective, as the majority of patients eventually become resistant to therapy. As chemoresistance is the leading cause of cancer spread, progression, and mortality, there is an increasing need for understanding the mechanisms by which cancer cells can evade therapy-induced cell death. The TAM (Tyro3, Axl, Mer) subfamily of RTKs in particular feature in a variety of cancer types that have developed resistance to a broad range of therapeutic agents, including both targeted as well as conventional chemotherapeutics. This article reviews the roles of TAMs as tumor drivers and as mediators of chemoresistance, and the potential effectiveness of targeting them as part of therapeutic strategies to delay or combat resistance. Cancer Res; 77(11); 2775-8. ©2017 AACR.

Gas6 Promotes Oligodendrogenesis and Myelination in the Adult Central Nervous System and After Lysolecithin-Induced Demyelination.

A key aim of therapy for multiple sclerosis (MS) is to promote the regeneration of oligodendrocytes and remyelination in the central nervous system (CNS). The present study provides evidence that the vitamin K-dependent protein growth arrest specific 6 (Gas6) promotes such repair in in vitro cultures of mouse optic nerve and cerebellum. We first determined expression of Gas6 and TAM (Tyro3, Axl, Mer) receptors in the mouse CNS, with all three TAM receptors increasing in expression through postnatal development, reaching maximal levels in the adult. Treatment of cultured mouse optic nerves with Gas6 resulted in significant increases in oligodendrocyte numbers as well as expression of myelin basic protein (MBP). Gas6 stimulation also resulted in activation of STAT3 in optic nerves as well as downregulation of multiple genes involved in MS development, including matrix metalloproteinase-9 (MMP9), which may decrease the integrity of the blood-brain barrier and is found upregulated in MS lesions. The cytoprotective effects of Gas6 were examined in in vitro mouse cerebellar slice cultures, where lysolecithin was used to induce demyelination. Cotreatment of cerebellar slices with Gas6 significantly attenuated demyelination as determined by MBP immunostaining, and Gas6 activated Tyro3 receptor through its phosphorylation. In conclusion, these results demonstrate that Gas6/TAM signaling stimulates the generation of oligodendrocytes and increased myelin production via Tyro3 receptor in the adult CNS, including repair after demyelinating injury. Furthermore, the effects of Gas6 on STAT3 signaling and matrix MMP9 downregulation indicate potential glial cell repair and immunoregulatory roles for Gas6, indicating that Gas6-TAM signaling could be a potential therapeutic target in MS and other neuropathologies.

Lycopene acts through inhibition of IκB kinase to suppress NF-κB signaling in human prostate and breast cancer cells.

We studied the effect of the potent dietary antioxidant lycopene on multiple points along the nuclear factor kappa B (NF-κB) signaling pathway in prostate and breast cancer cells. Lycopene significantly inhibited prostate and breast cancer cell growth at physiologically relevant concentrations of ≥1.25 µM. Similar concentrations also caused a 30-40 % reduction in inhibitor of kappa B (IκB) phosphorylation in the cells, as determined by western blotting. Furthermore, the same degree of inhibition by lycopene was observed for NF-κB transcriptional activity, as determined by reporter gene assay. Concomitant with this, immunofluorescence staining of lycopene-treated cells showed a significant suppression (≥25 %) of TNF-induced NF-κB p65 subunit nuclear translocation. Further probing of lycopene's effects on upstream elements of the NF-κB pathway showed a 25 % inhibition of both activity of recombinant IκB kinase ß (IKKß) kinase in a cell-free in vitro assay, as well as activity of IKKß immunoprecipitated from MDA-MB-231 cells treated with lycopene. In conclusion, the anticancer properties of lycopene may occur through inhibition of the NF-κB signaling pathway, beginning at the early stage of cytoplasmic IKK kinase activity, which then leads to reduced NF-κB-responsive gene regulation. Furthermore, these effects in cancer cells were observed at concentrations of lycopene that are relevant and achievable in vivo.

Hollow-layered nanoparticles for therapeutic delivery of peptide prepared using electrospraying.

The viability of single and coaxial electrospray techniques to encapsulate model peptide-angiotensin II into near mono-dispersed spherical, nanocarriers comprising N-octyl-O-sulphate chitosan and tristearin, respectively, was explored. The stability of peptide under controlled electric fields (during particle generation) was evaluated. Resulting nanocarriers were analysed using dynamic light scattering and electron microscopy. Cell toxicity assays were used to determine optimal peptide loading concentration (~1 mg/ml). A trout model was used to assess particle behaviour in vivo. A processing limit of 20 kV was determined. A range of electrosprayed nanoparticles were formed (between 100 and 300 nm) and these demonstrated encapsulation efficiencies of ~92 ± 1.8%. For the single needle process, particles were in matrix form and for the coaxial format particles demonstrated a clear core-shell encapsulation of peptide. The outcomes of in vitro experiments demonstrated triphasic activity. This included an initial slow activity period, followed by a rapid and finally a conventional diffusive phase. This was in contrast to results from in vivo cardiovascular activity in the trout model. The results are indicative of the substantial potential for single/coaxial electrospray techniques. The results also clearly indicate the need to investigate both in vitro and in vivo models for emerging drug delivery systems.

Small molecule inhibition of Axl receptor tyrosine kinase potently suppresses multiple malignant properties of glioma cells.

Glioblastoma multiforme (GBM) often features a combination of tumour suppressor gene inactivation and multiple oncogene overactivation. The Axl receptor tyrosine kinase is found overexpressed in GBM and thought to contribute to invasiveness, chemoresistance and poor survival. Here, we have evaluated the effect of BGB324, a clinical candidate Axl-specific small molecule inhibitor, on the invasive behaviour of human GBM cells in vitro, as an indicator of its potential in GBM therapy and also to elucidate the role of Axl in GBM pathogenesis.Two cultured adult GBM cell lines, SNB-19 and UP007, were treated with Gas6 and/or BGB324, and analysed in assays for survival, 3D colony growth, motility, migration and invasion. Western blot was used to detect protein expression and signal protein phosphorylation. In both cell lines, BGB324 inhibited specifically phosphorylation of Axl as well as Akt kinase further downstream. BGB324 also inhibited survival and proliferation of both cell lines in a concentration-dependent manner, as well as completely suppressing migration and invasion. Furthermore, our results indicate co-operative activation between the Axl and Tyro3 receptors, as well as ligand-independent Axl signalling, to take place in GBM cells. In conclusion, small molecule inhibitor-led targeting of Axl may be a promising therapy for GBM progression.

Functional characterisation of human cells harbouring a novel t(2p;7p) translocation involving TNS3 and EXOC6B genes.

BACKGROUND: Tensin3 is an intracellular cytoskeleton-regulating protein, the loss of which is associated with increased cell motility, as has been observed in some human cancers. A novel chromosomal translocation, t(2;7)(p13;p12), present in a patient with a complex syndromic phenotype, directly involves Tensin3 (TNS3) and EXOC6B genes. This translocation could impair the expression of Tensin3 and ExoC6B proteins, and potentially produce two novel fusion transcripts. In the present study, we have investigated the expression and phenotypic features of these potential products in cultured cells from the proband. METHODS: Skin fibroblasts isolated from the proband as well as an age-matched control were grown in cell culture. Cells were used for quantitative RT-PCR, western blot and immunofluorescent confocal microscopy, which determined Tensin3 gene and protein expression. Phase-contrast and confocal microscopy additionally revealed cellular phenotype differences. A scratch wound assay monitored by live cell imaging measured cellular migration rates. RESULTS: The levels of Tensin3 at both mRNA and protein levels were lower in proband cells versus control fibroblasts. Proband cells displayed broader and shorter morphologies versus control fibroblasts, and immunofluorescent staining revealed additional Tensin3 expression along cytoskeletal filaments and the cell periphery only in control fibroblasts. In addition, proband fibroblasts showed a significantly higher migration rate than control cells over 24 h. CONCLUSIONS: The phenotypic changes observed in proband cells may arise from TNS3 haploinsufficiency, causing partial loss of full-length Tensin3 protein. These results further expose a role for Tensin3 in cytoskeletal organisation and cell motility and may also help to explain the syndromic features observed in the patient.

CpG dinucleotide-specific hypermethylation of the TNS3 gene promoter in human renal cell carcinoma.

Tensin3 is a cytoskeletal regulatory protein that inhibits cell motility. Downregulation of the gene encoding Tensin3 (TNS3) in human renal cell carcinoma (RCC) may contribute to cancer cell metastatic behavior. We speculated that epigenetic mechanisms, e.g., gene promoter hypermethylation, might account for TNS3 downregulation. In this study, we identified and validated a TNS3 gene promoter containing a CpG island, and quantified the methylation level within this region in RCC. Using a luciferase reporter assay we demonstrated a functional minimal promoter activity for a 500-bp sequence within the TNS3 CpG island. Pyrosequencing enabled quantitative determination of DNA methylation of each CpG dinucleotide (a total of 43) in the TNS3 gene promoter. Across the entire analyzed CpG stretch, RCC DNA showed a higher methylation level than both non-tumor kidney DNA and normal control DNA. Out of all the CpGs analyzed, two CpG dinucleotides, specifically position 2 and 8, showed the most pronounced increases in methylation levels in tumor samples. Furthermore, CpG-specific higher methylation levels were correlated with lower TNS3 gene expression levels in RCC samples. In addition, pharmacological demethylation treatment of cultured kidney cells caused a 3-fold upregulation of Tensin3 expression. In conclusion, these results reveal a differential methylation pattern in the TNS3 promoter occurring in human RCC, suggesting an epigenetic mechanism for aberrant Tensin downregulation in human kidney cancer.