Dlk1 is a novel adrenocortical stem/progenitor cell marker that predicts malignancy in adrenocortical carcinoma.

Disruption of processes involved in tissue development and homeostatic self-renewal is increasingly implicated in cancer initiation, progression, and recurrence. The adrenal cortex is a dynamic tissue that undergoes life-long turnover. Here, using genetic fate mapping and murine adrenocortical carcinoma (ACC) models, we have identified a population of adrenocortical stem cells that express delta-like non-canonical Notch ligand 1 (DLK1). These cells are active during development, near dormant postnatally but are re-expressed in ACC. In a study of over 200 human ACC samples, we have shown DLK1 expression is ubiquitous and is an independent prognostic marker of recurrence-free survival. Paradoxically, despite its progenitor role, spatial transcriptomic analysis has identified DLK1 expressing cell populations to have increased steroidogenic potential in human ACC, a finding also observed in four human and one murine ACC cell lines. Finally, the cleavable DLK1 ectodomain is measurable in patients' serum and can discriminate between ACC and other adrenal pathologies with high sensitivity and specificity to aid in diagnosis and follow-up of ACC patients. These data demonstrate a prognostic role for DLK1 in ACC, detail its hierarchical expression in homeostasis and oncogenic transformation and propose a role for its use as a biomarker in this malignancy.

A Coxsackievirus B1-mediated nonlytic Extracellular Vesicle-to-cell mechanism of virus transmission and its possible control through modulation of EV release.

Like most non-enveloped viruses, CVB1 mainly uses cell lysis to spread. Details of a nonlytic virus transmission remain unclear. Extracellular Vesicles (EVs) transfer biomolecules between cells. We show that CVB1 entry into HeLa cells results in apoptosis and release of CVB1-induced 'medium-sized' EVs (CVB1i-mEVs). These mEVs (100-300 nm) harbour CVB1 as shown by immunoblotting with anti-CVB1-antibody; viral capsids were detected by transmission electron microscopy and RT-PCR revealed CVB1 RNA. The percentage of mEVs released from CVB1-infected HeLa cells harbouring virus was estimated from TEM at 34 %. Inhibition of CVB1i-mEV production, with calpeptin or siRNA knockdown of CAPNS1 in HeLa cells limited spread of CVB1 suggesting these vesicles disseminate CVB1 virions to new host cells by a nonlytic EV-to-cell mechanism. This was confirmed by detecting CVB1 virions inside HeLa cells after co-culture with CVB1i-mEVs; EV release may also prevent apoptosis of infected cells whilst spreading apoptosis to secondary sites of infection.

The crosstalk between FGF21 and GH leads to weakened GH receptor signaling and IGF1 expression and is associated with growth failure in very preterm infants.

Background: Fibroblast growth factor 21 (FGF21) is an essential metabolic regulator that adapts to changes in nutritional status. Severe childhood undernutrition induces elevated FGF21 levels, contributing to growth hormone (GH) resistance and subsequent linear growth attenuation potentially through a direct action on chondrocytes. Methods: In this study, we assessed expression of the components of both GH and FGF21 pathways in rare and unique human growth plates obtained from children. Moreover, we investigated the mechanistic interplay of FGF21 on GH receptor (GHR) signaling in a heterologous system. Results: Chronic FGF21 exposure increased GH-induced GHR turnover and SOCS2 expression, leading to the inhibition of STAT5 phosphorylation and IGF-1 expression. The clinical significance of FGF21 signaling through GH receptors was tested in nutritionally driven growth failure seen in very preterm (VPT) infants right after birth. VPT infants display an immediate linear growth failure after birth followed by growth catch-up. Consistent with the in vitro model data, we show that circulating FGF21 levels were elevated during deflection in linear growth compared to catch-up growth and were inversely correlated with the length velocity and circulating IGF1 levels. Conclusions: This study further supports a central role of FGF21 in GH resistance and linear growth failure and suggests a direct action on the growth plate.

Whole exome sequencing identifies deleterious rare variants in CCDC141 in familial self-limited delayed puberty.

Developmental abnormalities of the gonadotropin-releasing hormone (GnRH) neuronal network result in a range of conditions from idiopathic hypogonadotropic hypogonadism to self-limited delayed puberty. We aimed to discover important underlying regulators of self-limited delayed puberty through interrogation of GnRH pathways. Whole exome sequencing (WES) data consisting of 193 individuals, from 100 families with self-limited delayed puberty, was analysed using a virtual panel of genes related to GnRH development and function (n = 12). Five rare predicted deleterious variants in Coiled-Coil Domain Containing 141 (CCDC141) were identified in 21 individuals from 6 families (6% of the tested cohort). Homology modeling predicted all five variants to be deleterious. CCDC141 mutant proteins showed atypical subcellular localization associated with abnormal distribution of acetylated tubulin, and expression of mutants resulted in a significantly delayed cell migration, demonstrated in transfected HEK293 cells. These data identify mutations in CCDC141 as a frequent finding in patients with self-limited delayed puberty. The mis-localization of acetylated tubulin and reduced cell migration seen with mutant CCDC141 suggests a role of the CCDC141-microtubule axis in GnRH neuronal migration, with heterozygous defects potentially impacting the timing of puberty.

The role of delta-like non-canonical Notch ligand 1 (DLK1) in cancer.

Delta-like non-canonical Notch ligand 1 (DLK1) is a cleavable single-pass transmembrane protein and a member of the Notch/Delta/Serrate family. It is paternally expressed and belongs to a group of imprinted genes located on chromosome band 14q32 in humans and 12qF1 in mice. DLK1 is expressed in many human tissues during embryonic development but in adults expression is low and is mostly restricted to (neuro)endocrine tissues and other immature stem/progenitor cells (notably hepatoblasts). However, DLK1 is expressed at a high frequency in many common malignancies (liver, breast, brain, pancreas, colon and lung). More recently, high levels of expression have been identified in endocrine-related cancers such as ovarian and adrenocortical carcinoma. There is growing evidence that DLK1 expression in cancer is associated with worse prognosis and that DLK1 may be a marker of cancer stem cells. Although the exact mechanism through which DLK1 functions is not fully understood, it is known to maintain cells in an undifferentiated phenotype and has oncogenic properties. These effects are partly exacted through interaction with the Notch signalling pathway. In this review, we have detailed the functional role of DLK1 within physiology and malignancy and posited a mechanism for how it exacts its oncogenic effects. In describing the expression of DLK1 in cancer and in healthy tissue, we have highlighted the potential for its use both as a biomarker and as a potential therapeutic target.

Towards novel treatments for adrenal diseases: Cell- and gene therapy-based approaches.

Adrenal insufficiency, the inability to produce adequate levels of corticosteroids, is a multi-causal disease that requires lifelong daily hormone replacement. Nevertheless, this cannot replace the physiological demand for steroids which are secreted following a circadian rhythm and vary in periods of stress; the consequences of under- or over-replacement include adrenal crisis and metabolic disturbances, respectively. Although clinical research has focused on enhancing the effectiveness/reducing side effects of current treatment modalities, only small improvements are deemed possible; thus, alternative solutions are urgently needed. Gene and cell therapy strategies have opened new possibilities for the cure of many diseases in a way that has never been possible before and could offer a viable option for the cure of adrenal diseases. The current state of cell- and gene-based approaches to restore adrenocortical function is discussed in this review.

Stem Cells, Self-Renewal, and Lineage Commitment in the Endocrine System.

The endocrine system coordinates a wide array of body functions mainly through secretion of hormones and their actions on target tissues. Over the last decades, a collective effort between developmental biologists, geneticists, and stem cell biologists has generated a wealth of knowledge related to the contribution of stem/progenitor cells to both organogenesis and self-renewal of endocrine organs. This review provides an up-to-date and comprehensive overview of the role of tissue stem cells in the development and self-renewal of endocrine organs. Pathways governing crucial steps in both development and stemness maintenance, and that are known to be frequently altered in a wide array of endocrine disorders, including cancer, are also described. Crucially, this plethora of information is being channeled into the development of potential new cell-based treatment modalities for endocrine-related illnesses, some of which have made it through clinical trials.

DLK1/PREF1 marks a novel cell population in the human adrenal cortex.

The adrenal cortex governs fundamental metabolic processes though synthesis of glucocorticoid, mineralocorticoids and androgens. Studies in rodents have demonstrated that the cortex undergoes a self-renewal process and that capsular/subcapsular stem/progenitor cell pools differentiate towards functional steroidogenic cells supporting the dynamic centripetal streaming of adrenocortical cells throughout life. We previously demonstrated that the Notch atypical ligand Delta-like homologue 1 (DLK1)/preadipocyte factor 1 (PREF1) is expressed in subcapsular Sf1 and Shh-positive, CYP11B1-negative and CYP11B2-partially positive cortical progenitor cells in rat adrenals, and that secreted DLK1 can modulate GLI1 expression in H295R cells. Here we show that the human adrenal cortex remodels with age to generate clusters of relatively undifferentiated cells expressing DLK1. These clusters (named DLK1-expressing cell clusters or DCCs) increased with age in size and were found to be different entities to aldosterone-producing cell clusters, another well-characterized and age-dependent cluster structure. DLK1 was markedly overexpressed in adrenocortical carcinomas but not in aldosterone-producing adenomas. Thus, this data identifies a novel cell population in the human adrenal cortex and might suggest a yet-to be identified role of DLK1 in the pathogenesis of adrenocortical carcinoma in humans.

Phosphorylation of protein kinase B, the key enzyme in insulin-signaling cascade, is enhanced in linoleic and arachidonic acid-treated HT29 and HepG2 cells.

OBJECTIVES: Defects in the insulin-signaling pathway have been implicated in the pathogenesis of impaired glucose uptake, insulin resistance, and type 2 diabetes. However, the specific defects that precipitate these abnormalities are yet to be fully elucidated. After binding to insulin, the plasma membrane-embedded insulin receptor transmembrane protein initiates a cascade of phosphorylation that leads to the activation of protein kinase B (AKT) and subsequently to the initiation of some metabolic actions of insulin. The activities of this receptor, insulin binding, and tyrosine kinase activation is dependent on its plasma lipid environment. Published data on the influence of omega-3 and -6 polyunsaturated fatty acids on insulin response are scarce. Moreover, the findings of the published investigations, most of which used omega-3 and -6, polyunsaturated fatty-acid blends, have been inconclusive. Hence, further, well thought out research is needed. The aim of the current study was to elucidate the effect of treatments with linoleic acid (LNA), arachidonic acid (ARA), alpha-linolenic acid (ALA), docoshexaenoic acid (DHA), and eicosapentaenoic acid (EPA) on cell membrane composition and consequently on the insulin-signaling pathway and specifically AKT phosphorylation. METHODS: Human colon adenocarcinoma (HT29) and liver hepatocellular (HepG2) cells were treated with or without 40 µM of LNA, ARA, ALA, EPA, or DHA for 48 h, the fatty-acid composition of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) from the treated cells by capillary gas liquid chromatograph. Cells were incubated for 30 min with or without human insulin (50 ng/mL), and the phosphorylation of AKT was assessed with the use of Western blotting. RESULTS: The fatty acids were incorporated in the PtdCho and PtdEtn of both cell lines, but the level of incorporation was higher in HT29. Phosphorylation of AKT increased when HT29 was treated with LNA (P < 0.05) and ARA (P < 0.01) but not with ALA, EPA, or DHA. A similar but non-significant increase in AKT phosphorylation was observed in LNA- and ARA- treated HepG2 cells. CONCLUSIONS: The finding of this investigation demonstrates that plasma membrane lipid bilayer enrichment with LNA or ARA treatment enhances insulin action by AKT activation.

Omega 3 (n-3) fatty acids down-regulate nuclear factor-kappa B (NF-κB) gene and blood cell adhesion molecule expression in patients with homozygous sickle cell disease.

Chronic inflammation and reduced blood levels of omega-3 fatty acids (n-3) are known characteristics of sickle cell disease (SCD).The anti-inflammatory properties of n-3 fatty acids are well recognized. Omega-3 treated (n = 24), hydroxyurea (HU) treated (n = 18), and n-3 untreated (n=21) homozygous SCD patients (HbSS) and healthy (HbAA) controls (n = 25) matched for age (5-16 years), gender and socioeconomic status were studied. According to age (5-10) or (11-16) years, two or three capsules containing 277.8 mg docosahexaenoic (DHA) and 39.0mg eicosapentaenoic (EPA) or high oleic acid placebo (41%) were assigned to n-3 treated and n-3 untreated groups, respectively. Hydroxyurea treated group was on dosage more than 20 mg/kg/day. The effect of supplementation on systemic and blood cell markers of inflammation was investigated. The n-3 treated group had higher levels of DHA and EPA (p < 0.001) and lower white blood cell count and monocyte integrin (p < 0.05) compared with the n-3 untreated. No difference was detected between the two groups regarding C-reactive protein, granulocytes integrin and selectin, plasma tumour necrosis factor-α and interleukin-10. The n-3 treated group had lowered nuclear factor-kappa B (NF-κB) gene expression compared to n-3 untreated and HU treated groups (p < 0.05). This study provides evidence that supplementation with n-3 fatty acids may ameliorate inflammation and blood cell adhesion in patients with SCD.

Docosahexaenoic and eicosapentaenoic acid supplementation does not exacerbate oxidative stress or intravascular haemolysis in homozygous sickle cell patients.

We investigated whether or not Omega-3 long-chain polyunsaturated fatty acid (omega-3 LCPUFA) supplementation exacerbates oxidative stress in homozygous sickle cell patients aged 2 to 14 years. Depending on their age, they received between one and three omega-3 (277.8mg DHA and 39.0mg EPA/capsule) or placebo (high oleic acid sunflower seed oil) capsules for one year. Supplementation increased significantly the levels of the two fatty acids in red cell phosphatidylcholine and phosphatidylethanolamine (p<0.001). The patients who received omega-3 LCPUFA compared with their placebo-taking counterparts had a higher concentration of plasma vitamin E at one year (14.3±2.8 versus 12.3±2.8µmol/l; p<0.001). The two groups had comparable concentrations of the vitamin at six month intervention (10.8±2.2 versus 10.7±2.9µmol/l; p>0.05) and baseline (10.7±3.1 versus 10.7±2.8µmol/l; p>0.05). After six month of intervention, the patients on omega 3 fatty acids had lower GPx-1 (33.5±13.4 versus 46.6 ±17.6, p<0.01) and Cu/Zn-SOD (1070±600 versus 1470±690 p<0.05) activities than at baseline. GPx-1 (33.5±17.6IU/g Hb versus 43.7±13.2IU/g Hb; p<0.01) and Cu/Zn-SOD (1070±600IU/g Hb versus 1360±920IU/g Hb; p>0.05) activities were reduced in the omega 3 compared with the placebo at six month intervention. There was no difference in the activity of either of the enzymes between baseline and six month intervention in the placebo group (p>0.05). This study demonstrates; DHA and EPA supplementation, rather than exacerbating the inherent oxidative stress associated with the disease, seems to provide an antioxidant protection. Hence, it will be safe to provide omega-3 LCPUFA to sickle cell patients to help ameliorate vaso-occlusive and haemolytic crises and membrane fatty acid abnormality.