dsRNAi-mediated silencing of PIAS2beta specifically kills anaplastic carcinomas by mitotic catastrophe.

The E3 SUMO ligase PIAS2 is expressed at high levels in differentiated papillary thyroid carcinomas but at low levels in anaplastic thyroid carcinomas (ATC), an undifferentiated cancer with high mortality. We show here that depletion of the PIAS2 beta isoform with a transcribed double-stranded RNA-directed RNA interference (PIAS2b-dsRNAi) specifically inhibits growth of ATC cell lines and patient primary cultures in vitro and of orthotopic patient-derived xenografts (oPDX) in vivo. Critically, PIAS2b-dsRNAi does not affect growth of normal or non-anaplastic thyroid tumor cultures (differentiated carcinoma, benign lesions) or cell lines. PIAS2b-dsRNAi also has an anti-cancer effect on other anaplastic human cancers (pancreas, lung, and gastric). Mechanistically, PIAS2b is required for proper mitotic spindle and centrosome assembly, and it is a dosage-sensitive protein in ATC. PIAS2b depletion promotes mitotic catastrophe at prophase. High-throughput proteomics reveals the proteasome (PSMC5) and spindle cytoskeleton (TUBB3) to be direct targets of PIAS2b SUMOylation at mitotic initiation. These results identify PIAS2b-dsRNAi as a promising therapy for ATC and other aggressive anaplastic carcinomas.

RET signalling provides tumorigenic mechanism and tissue specificity for AIP-related somatotrophinomas.

It is unclear how loss-of-function germline mutations in the widely-expressed co-chaperone AIP, result in young-onset growth hormone secreting pituitary tumours. The RET receptor, uniquely co-expressed in somatotrophs with PIT1, induces apoptosis when unliganded, while RET supports cell survival when it is bound to its ligand. We demonstrate that at the plasma membrane, AIP is required to form a complex with monomeric-intracellular-RET, caspase-3 and PKCδ resulting in PIT1/CDKN2A-ARF/p53-apoptosis pathway activation. AIP-deficiency blocks RET/caspase-3/PKCδ activation preventing PIT1 accumulation and apoptosis. The presence or lack of the inhibitory effect on RET-induced apoptosis separated pathogenic AIP variants from non-pathogenic ones. We used virogenomics in neonatal rats to demonstrate the effect of mutant AIP protein on the RET apoptotic pathway in vivo. In adult male rats altered AIP induces elevated IGF-1 and gigantism, with pituitary hyperplasia through blocking the RET-apoptotic pathway. In females, pituitary hyperplasia is induced but IGF-1 rise and gigantism are blunted by puberty. Somatotroph adenomas from pituitary-specific Aip-knockout mice overexpress the RET-ligand GDNF, therefore, upregulating the survival pathway. Somatotroph adenomas from patients with or without AIP mutation abundantly express GDNF, but AIP-mutated tissues have less CDKN2A-ARF expression. Our findings explain the tissue-specific mechanism of AIP-induced somatotrophinomas and provide a previously unknown tumorigenic mechanism, opening treatment avenues for AIP-related tumours.

Synthesis and In Vivo Evaluation of Insulin-Loaded Whey Beads as an Oral Peptide Delivery System.

For many diabetics, daily, lifelong insulin injections are required to effectively manage blood glucose levels and the complications associated with the disease. This can be a burden and reduces patient quality of life. Our goal was to develop a more convenient oral delivery system that may be suitable for insulin and other peptides. Insulin was entrapped in 1.5-mm beads made from denatured whey protein isolate (dWPI) using gelation. Beads were then air-dried with fumed silica, Aerosil®. The encapsulation efficiency was ~61% and the insulin loading was ~25 µg/mg. Dissolution in simulated gastric-, and simulated intestinal fluids (SGF, SIF) showed that ~50% of the insulin was released from beads in SGF, followed by an additional ~10% release in SIF. The omission of Aerosil® allowed greater insulin release, suggesting that it formed a barrier on the bead surface. Circular dichroism analysis of bead-released insulin revealed an unaltered secondary structure, and insulin bioactivity was retained in HepG2 cells transfected to assess activation of the endogenous insulin receptors. Insulin-entrapped beads were found to provide partial protection against pancreatin for at least 60 min. A prototype bead construct was then synthesised using an encapsulator system and tested in vivo using a rat intestinal instillation bioassay. It was found that 50 IU/kg of entrapped insulin reduced plasma glucose levels by 55% in 60 min, similar to that induced by subcutaneously (s.c.)-administered insulin (1 IU/kg). The instilled insulin-entrapped beads produced a relative bioavailability of 2.2%. In conclusion, when optimised, dWPI-based beads may have potential as an oral peptide delivery system.

A nanoemulsion/micelles mixed nanosystem for the oral administration of hydrophobically modified insulin.

The potential of nanoemulsions for the oral administration of peptides is still in its early stage. The aim of the present work was to rationally design, develop, and fully characterize a new nanoemulsion (NE) intended for the oral administration of hydrophobically modified insulin (HM-insulin). Specific components of the NE were selected based on their enhancing permeation properties as well as their ability to improve insulin association efficiency (Miglyol 812, sodium taurocholate), stability in the intestinal fluids, and mucodiffusion (PEGylated phospholipids and poloxamer 407). The results showed that the NE co-existed with a population of micelles, forming a mixed system that exhibited a 100% of HM-insulin association efficiency. The nanosystem showed good stability and miscibility in different bio-relevant media and displayed an acceptable mucodiffusive behavior in porcine mucus. In addition, it exhibited a high interaction with cell mono-cultures (Caco -2 and C2BBe1 human colon carcinoma Caco-2 clone cells) and co-cultures (C2BBe1 human colon carcinoma Caco-2 clone/HT29-MTX cells). The internalization in Caco-2 monolayers was also confirmed by confocal microscopy. Finally, the promising in vitro behavior of the nanosystem in terms of overcoming the biological barriers of the intestinal tract was translated into a moderate, although significant, hypoglycemic response (≈ 20-30%), following intestinal administration to both healthy and diabetic rat models. Overall, this information underlines the crucial steps to address when designing peptide-based nanoformulations to successfully overcome the intestinal barriers associated to the oral modality of administration.

Sequential Colocalization of ERa, PR, and AR Hormone Receptors Using Confocal Microscopy Enables New Insights into Normal Breast and Prostate Tissue and Cancers.

Multiplex immunohistochemistry (mIHC) use markers staining different cell populations applying widefield optical microscopy. Resolution is low not resolving subcellular co-localization. We sought to colocalize markers at subcellular level with antibodies validated for clinical diagnosis, including the single secondary antibody (combination of anti-rabbit/mouse-antibodies) used for diagnostic IHC with any primary antibody, and confocal microscopy. We explore colocalization in the nucleus (ColNu) of nuclear hormone receptors (ERa, PR, and AR) along with the baseline marker p63 in paired samples of breast and prostate tissues. We established ColNu mIHCF as a reliable technique easily implemented in a hospital setting. In ERa+ breast cancer, we identified different colocalization patterns (nuclear or cytoplasmatic) with PR and AR on the luminal epithelium. A triple-negative breast-cancer case expressed membrane-only ERa. A PR-only case was double positive PR/p63. In normal prostate, we identified an ERa+/p63+/AR-negative distinct population. All prostate cancer cases characteristically expressed ERa on the apical membrane of the AR+ epithelium. We confirmed this using ERa IHC and needle-core biopsies. ColNu mIHCF is feasible and already revealed a new marker for prostate cancer and identified sub-patterns in breast cancer. It could be useful for pathology as well as for functional studies in normal prostate and breast tissues.

GFRα 1-2-3-4 co-receptors for RET Are co-expressed in Pituitary Stem Cells but Individually Retained in Some Adenopituitary Cells.

The RET tyrosine kinase receptor is expressed by the endocrine somatotroph cells of the pituitary where it has important functions regulating survival/apoptosis. However, RET is also expressed by the GPS pituitary stem cells localized in a niche between the adenopituitary and the intermediate lobe. To bind any of its four ligands, RET needs one of four co-receptors called GFRα1-4. It has been previously shown that GFRα1 is expressed by somatotroph cells and acromegaly tumors. GFRα2 was shown to be expressed by pituitary stem cells. GFRα4 was proposed as not expressed in the pituitary. Here we study the RNA and protein expression of the four GFRα co-receptors for RET in rat and human pituitary. The four co-receptors were abundantly expressed at the RNA level both in rat and human pituitary, although GFRα4 was the less abundant. Multiple immunofluorescence for each co-receptor and ß-catenin, a marker of stem cell niche was performed. The four GFRα co-receptors were co-expressed by the GPS cells at the niche colocalizing with ß-catenin. Isolated individual scattered cells positive for one or other receptor could be found through the adenopituitary with low ß-catenin expression. Some of them co-express GFRα1 and PIT1. Immunohistochemistry in normal human pituitary confirmed the data. Our data suggest that the redundancy of GFRα co-expression is a self-supportive mechanism which ensures niche maintenance and proper differentiation.

Mechanistic investigations into the encapsulation and release of small molecules and proteins from a supramolecular nucleoside gel in vitro and in vivo.

Supramolecular gels have recently emerged as promising biomaterials for the delivery of a wide range of bioactive molecules, from small hydrophobic drugs to large biomolecules such as proteins. Although it has been demonstrated that each encapsulated molecule has a different release profile from the hydrogel, so far diffusion and steric impediment have been identified as the only mechanisms for the release of molecules from supramolecular gels. Erosion of a supramolecular gel has not yet been reported to contribute to the release profiles of encapsulated molecules. Here, we use a novel nucleoside-based supramolecular gel as a drug delivery system for proteins with different properties and a hydrophobic dye and describe for the first time how these materials interact, encapsulate and eventually release bioactive molecules through an erosion-based process. Through fluorescence microscopy and spectroscopy as well as small angle X-ray scattering, we show that the encapsulated molecules directly interact with the hydrogel fibres - rather than being physically entrapped in the gel network. The ability of these materials to protect proteins against enzymatic degradation is also demonstrated here for the first time. In addition, the released proteins were proven to be functional in vitro. Real-time fluorescence microscopy together with macroscopic release studies confirm that erosion is the key release mechanism. In vivo, the gel completely degrades after two weeks and no signs of inflammation are detected, demonstrating its in vivo safety. By establishing the contribution of erosion as a key driving force behind the release of bioactive molecules from supramolecular gels, this work provides mechanistic insight into the way molecules with different properties are encapsulated and released from a nucleoside-based supramolecular gel and sets the basis for the design of more tailored supramolecular gels for drug delivery applications.

Unmasking a new prognostic marker and therapeutic target from the GDNF-RET/PIT1/p14ARF/p53 pathway in acromegaly.

BACKGROUND: Acromegaly is produced by excess growth hormone secreted by a pituitary adenoma of somatotroph cells (ACRO). First-line therapy, surgery and adjuvant therapy with somatostatin analogs, fails in 25% of patients. There is no predictive factor of resistance to therapy. New therapies are investigated using few dispersed tumor cells in acute primary cultures in standard conditions where the cells do not grow, or using rat pituitary cell lines that do not maintain the full somatotroph phenotype. The RET/PIT1/p14ARF/p53 pathway regulates apoptosis in normal pituitary somatotrophs whereas the RET/GDNF pathway regulates survival, controlling PIT1 levels and blocking p14ARF (ARF) and p53 expression. METHODS: We investigated these two RET pathways in a prospective series of 32 ACRO and 63 non-functioning pituitary adenomas (NFPA), studying quantitative RNA and protein gene expression for molecular-clinical correlations and how the RET pathway might be implicated in therapeutic success. Clinical data was collected during post-surgical follow-up. We also established new'humanized' pituitary cultures, allowing 20 repeated passages and maintaining the pituitary secretory phenotype, and tested five multikinase inhibitors (TKI: Vandetanib, Lenvatinib, Sunitinib, Cabozantinib and Sorafenib) potentially able to act on the GDNF-induced RET dimerization/survival pathway. Antibody arrays investigated intracellular molecular pathways. FINDINGS: In ACRO, there was specific enrichment of all genes in both RET pathways, especially GDNF. ARF and GFRA4 gene expression were found to be opposing predictors of response to first-line therapy. ARF cut-off levels, calculated categorizing by GNAS mutation, were predictive of good response (above) or resistance (below) to therapy months later. Sorafenib, through AMPK, blocked the GDNF/AKT survival action without altering the RET apoptotic pathway. INTERPRETATION: Tumor ARF mRNA expression measured at the time of the surgery is a prognosis factor in acromegaly. The RET inhibitor, Sorafenib, is proposed as a potential treatment for resistant ACRO. FUND: This project was supported by national grants from Agencia Estatal de Investigación (AEI) and Instituto Investigación Carlos III, with participation of European FEDER funds, to IB (PI150056) and CVA (BFU2016-76973-R). It was also supported initially by a grant from the Investigator Initiated Research (IIR) Program (WI177773) and by a non-restricted Research Grant from Pfizer Foundation to IB. Some of the pituitary acromegaly samples were collected in the framework of the Spanish National Registry of Acromegaly (REMAH), partially supported by an unrestricted grant from Novartis to the Spanish Endocrine Association (SEEN). CVA is also supported from a grant of Medical Research Council UK MR/M018539/1.

Protamine nanocapsules as carriers for oral peptide delivery.

Peptides represent a promising therapeutic class with the potential to alleviate many severe diseases. A key limitation of these active molecules relies on the difficulties for their efficient oral administration. The objective of this work has been the rational design of polymer nanocapsules (NCs) intended for the oral delivery of peptide drugs. For this purpose, we selected insulin glulisine as a model peptide. The polymer shell of the NCs was made of a single layer of protamine, a cationic polypeptide selected for its cell penetration properties, or a double protamine/polysialic acid (PSA) layer. Insulin glulisine-loaded protamine and protamine/PSA NCs, prepared by the solvent displacement method, exhibited a size that varied in the range of 200-400 nm and a neutral surface charge (from +8 mV to -6 mV), depending on the formulation. The stability of the encapsulated peptide was assessed using circular dichroism and an in vitro cell activity study. Colloidal stability studies were also performed in simulated intestinal media containing enzymes and the results indicated that protamine NCs were stable and able to protect insulin from the harsh intestinal environment, and that this capacity could be further enhanced with a double PSA-Protamine layer. These NCs were freeze-dried and stored at room temperature without alteration of the physicochemical properties. When the insulin-loaded protamine NCs were administered intra-intestinally to diabetic rats (12 h fasting) it resulted in a prolonged glucose reduction (60%) as compared to the control insulin solution. This work raises prospects that protamine NCs may have a potential as oral peptide delivery nanocarriers.