The tumour-derived extracellular vesicle proteome varies by endometrial cancer histology and is confounded by an obesogenic environment.

Endometrial cancer, the most common gynaecological cancer worldwide, is closely linked to obesity and metabolic diseases, particularly in younger women. New circulating biomarkers have the potential to improve diagnosis and treatment selections, which could significantly improve outcomes. Our approach focuses on extracellular vesicle (EV) biomarker discovery by directly profiling the proteome of EVs enriched from frozen biobanked endometrial tumours. We analysed nine tissue samples to compare three clinical subgroups-low BMI (Body Mass Index) Endometrioid, high BMI Endometrioid, and Serous (any BMI)-identifying proteins related to histological subtype, BMI, and shared secreted proteins. Using collagenase digestion and size exclusion chromatography, we successfully enriched generous quantities of EVs (range 204.8-1291.0 µg protein: 1.38 × 1011-1.10 × 1012 particles), characterised by their size (∼150 nm), expression of EV markers (CD63/81), and proposed endometrial cancer markers (L1CAM, ANXA2). Mass spectrometry-based proteomic profiling identified 2075 proteins present in at least one of the 18 samples. Compared to cell lysates, EVs were successfully depleted for mitochondrial and blood proteins and enriched for common EV markers and large secreted proteins. Further analysis highlighted significant differences in EV protein profiles between the high BMI subgroup and others, underlining the impact of comorbidities on the EV secretome. Interestingly, proteins differentially abundant in tissue subgroups were largely not also differential in matched EVs. This research identified secreted proteins known to be involved in endometrial cancer pathophysiology and proposed novel diagnostic biomarkers (EIF6, MUC16, PROM1, SLC26A2).

Novel cationic carotenoid lipids as delivery vectors of antisense oligonucleotides for exon skipping in Duchenne muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is a common, inherited, incurable, fatal muscle wasting disease caused by deletions that disrupt the reading frame of the DMD gene such that no functional dystrophin protein is produced. Antisense oligonucleotide (AO)-directed exon skipping restores the reading frame of the DMD gene, and truncated, yet functional dystrophin protein is expressed. The aim of this study was to assess the efficiency of two novel rigid, cationic carotenoid lipids, C30-20 and C20-20, in the delivery of a phosphorodiamidate morpholino (PMO) AO, specifically designed for the targeted skipping of exon 45 of DMD mRNA in normal human skeletal muscle primary cells (hSkMCs). The cationic carotenoid lipid/PMO-AO lipoplexes yielded significant exon 45 skipping relative to a known commercial lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC).

Delivery of therapeutic proteins as secretable TAT fusion products.

The trans-acting activator of transcription (TAT) protein transduction domain (PTD) mediates the transduction of peptides and proteins into target cells. The TAT-PTD has an important potential as a tool for the delivery of therapeutic agents. The production of TAT fusion proteins in bacteria, however, is problematic because of protein insolubility and the absence of eukaryotic post-translational modification. An attractive alternative, both for in vitro protein production and for in vivo applications, is the use of higher eukaryotic cells for secretion of TAT fusion proteins. However, the ubiquitous expression of furin endoprotease (PACE or SPC1) in the Golgi/endoplasmic reticulum, and the presence of furin recognition sequences within TAT-PTD, results in the cleavage and loss of the TAT-PTD domain during its secretory transition through the endoplasmic reticulum and Golgi. In this study, we show the development of a synthetic TATkappa-PTD in which mutation of the furin recognition sequences, but retention of protein transduction activity, allows secretion of recombinant proteins, followed by successful uptake of the modified protein, by the target cells. This system was used to successfully secrete marker protein, green fluorescent protein (GFP), and apoptin, a protein with tumor-specific cytotoxicity. Detection of GFP, phosphorylation, and induction of cell death by TATkappa-GFP-apoptin indicated that the secreted proteins were functional in target cells. This novel strategy therefore has important potential for the efficient delivery of therapeutic proteins.

E1A-mediated suppression of EGFR expression and induction of apoptosis in head and neck squamous carcinoma cell lines.

Previous studies have shown early region 1A (E1A) gene to inhibit the proliferation of tumour cells with wild-type, but not mutant, p53. E1A has also been shown to downregulate c-erb-B-2/neu expression, resulting in inhibition of growth in c-erb-B-2/neu overexpressing tumour cells. In this study, we have investigated the effect of E1A expression on four head and neck squamous cell carcinoma (HNSCC) cell lines that do not overexpress c-erb-B-2/neu. Cell cycle and Western blot analysis show E1A-mediated induction of apoptosis in all cell lines examined. This induction of apoptosis was independent of the p53 status as it occurred in the cell lines with wild-type, mutated or deleted p53. However, there was no evidence of E1A-induced apoptosis in a p53(+ve) normal human fibroblast cell line, 1BR3. Analysis of apoptosis in the SCC cell lines demonstrated E1A-mediated downregulation of EGFR, which was overexpressed in each of these cell lines. Overexpression of an exogenously introduced EGFR, under the control of an E1A-insensitive heterologous promoter, blocked E1A induction of apoptosis in these cells. Therefore, E1A-mediated downregulation of EGFR expression appears to be the cause, rather than a consequence of E1A-induced apoptosis in these SCC cell lines. Previous studies have shown downregulation of EGFR expression by PML. Interestingly, E1A expression in the HNSCC cells altered the pattern of PML distribution and induced the level of PML protein, thus suggesting that E1A-mediated downregulation of EGFR may occur via direct or indirect interactions with PML. These findings demonstrate a novel pathway by which E1A can induce apoptosis and identify EGFR as a potential target for the development of therapeutic strategies against epithelial malignancies, the majority of which have abnormal EGFR expression.

Novel cationic polyene glycol phospholipids as DNA transfer reagents--lack of a structure-activity relationship due to uncontrolled self-assembling processes.

Cationic glycol phospholipids were synthesized introducing chromophoric, rigid polyenoic C20:5 and C30:9 chains next to saturated flexible alkyl chains of variable lengths C6-20:0. Surface properties and liposome formation of the amphiphilic compounds were determined, the properties of liposome/DNA complexes (lipoplexes) were established using three formulations (no co-lipid, DOPE as a co-lipid, or cholesterol as a co-lipid), and the microstructure of the best transfecting compounds inspected using small angle X-ray diffraction to explore details of the partially ordered structures of the systems that constitute the series. Transfection and cytotoxicity of the lipoplexes were evaluated by DNA delivery to Chinese hamster ovary (CHO-K1) cells using the cationic glycerol phospholipid 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC) as a reference compound. The uncontrollable self-association of the molecules in water resulted in aggregates and liposomes of quite different sizes without a structure-property relationship. Likewise, adding DNA to the liposomes gave rise to unpredictable sized lipoplexes, which, again, transfected without a structure-activity relationship. Nevertheless, one compound among the novel lipids (C30:9 chain paired with a C20:0 chain) exhibited comparable transfection efficiency and toxicity to the control cationic lipid EPC. Thus, the presence of a rigid polyene chain in this best performing achiral glycol lipid did not have an influence on transfection compared with the chiral glycerolipid reference ethyl phosphocholine EPC with two flexible saturated C14 chains.

Specific isoforms of p73 control the induction of cell death induced by the viral proteins, E1A or apoptin.

A member of the p53 family, p73, has several isoforms and differentially regulates transcription of genes involved in the control of the cell cycle and apoptosis. We have previously shown efficient and p53-independent, tumor-specific cell death induced by the viral proteins E1A and Apoptin. Here, we demonstrate that the induction of apoptosis by these viral proteins involves activation of TAp73. Both E1A and Apoptin induced expression of endogenous TAp73 and the p53/p73 BH3-only pro-apoptotic target, PUMA, independently of the p53 function. Furthermore, exogenous expression of TAp73 isoforms, particularly TAp73beta, sensitized cells to killing by both E1A and Apoptin, while expression of DeltaNp73alpha blocked this activity. Besides, knockout of the p73 regulator, c-Abl, attenuated E1A-induced apoptosis. In accordance with the role of p73 in apoptosis induced by these viral proteins, overexpression of TAp73beta strongly induced apoptosis in p53-deficient cancer cells in vitro and in HNSCC xenografts. Using a doxycycline-inducible system, we provide evidence for target selectivity and significant differences in protein stability for specific p73 isoforms, suggesting a diverse and pivotal role for p73 in response to various genotoxic agents. Collectively, our data show that in the absence of the p53 function, viral proteins E1A and Apoptin utilize the p73 pathway to induce efficient tumor cell death.

E1A activates transcription of p73 and Noxa to induce apoptosis.

p73, a member of the p53 family of proteins, transcriptionally activates a number of genes involved in the control of cell cycle and apoptosis. Overexpression of p73 was detected in a large number of primary head and neck cancers, and in the established cell lines examined, these all contained inactivating p53 mutations. The significance of p73 overexpression in the pathogenesis of head and neck cancer is currently unclear. We have shown that the expression of adenovirus 5 E1A in a panel of head and neck cancer cell lines induces apoptosis independently of their p53 status. In this study we examined the role of p73 and its transcriptional targets in E1A-mediated induction of apoptosis. E1A expression resulted in significant activation of the TAp73 promoter but had no effect on the alternative, DeltaNp73 promoter. E1A also increased expression of endogenous TAp73 mRNA and protein. E1A mutants lacking the p300- and/or pRB-binding sites showed reduced ability to activate the TAp73 promoter. Additionally, mutations in the E2F1-binding sites in the TAp73 promoter impaired activation by E1A. Importantly, expression of the 13S isoform of E1A substantially induced the p53 apoptotic target Noxa in several p53-deficient cancer cell lines. Our results indicate that E1A activation of p73 and the p53 apoptotic target Noxa can occur in the absence of a functional p53. This activation is likely to play a key role in the mechanism of p53-independent apoptosis induced by E1A in some cancers and may provide an avenue for future cancer therapies.