The multiple functions of miR-574-5p in the neuroblastoma tumor microenvironment

Neuroblastoma is the most common extracranial solid tumor in childhood and arises from neural crest cells of the developing sympathetic nervous system. Prostaglandin E2 (PGE2) has been identified as a key pro-inflammatory mediator of the tumor microenvironment (TME) that promotes neuroblastoma progression. We report that the interaction between the microRNA miR-574-5p and CUG-binding protein 1 (CUGBP1) induces the expression of microsomal prostaglandin E2 synthase 1 (mPGES-1) in neuroblastoma cells, which contributes to PGE2 biosynthesis. PGE2 in turn specifically induces the sorting of miR-574-5p into small extracellular vesicles (sEV) in neuroblastoma cell lines. sEV are one of the major players in intercellular communication in the TME. We found that sEV-derived miR-574-5p has a paracrine function in neuroblastoma. It acts as a direct Toll-like receptor 7/8 (TLR7/8) ligand and induces α-smooth muscle actin (α-SMA) expression in fibroblasts, contributing to fibroblast differentiation. This is particularly noteworthy as it has an opposite function to that in the TME of lung carcinoma, another PGE2 dependent tumor type. Here, sEV-derived miR-574-5p has an autokrine function that inhibits PGE2 biosynthesis in lung cancer cells. We report that the tetraspanin composition on the surface of sEV is associated with the function of sEV-derived miR-574-5p. This suggests that the vesicles do not only transport miRs, but also appear to influence their mode of action

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Small extracellular vesicle-derived miR-574-5p regulates PGE₂ biosynthesis via TLR7/8 in lung cancer

Lung cancer (LC) is the leading cause of cancer-related deaths. Its progression is strongly enhanced by intercellular communication in the tumor microenvironment (TME). One of the main players in intercellular communication is small extracellular vesicles (sEV). sEV transport various biological cargoes such as microRNAs (miRs), a class of small non-coding RNAs, and thus trigger diverse responses in recipient cells. In addition to sEV, inflammatory processes promote tumor progression. A key inflammatory mediator in LC is prostaglandin E2 (PGE2). It highly contributes to inflammation, angiogenesis, immune evasion, and proliferation in the TME. To date, a possible relationship between inflammatory mediators like PGE2 and miR content in sEV remains unknown. Hence, this study aimed to analyze the impact of PGE2 on miR sorting into sEV. In this context, special attention was paid to miR-574-5p because it was shown to regulate PGE2 biosynthesis in lung adenocarcinoma (AC) cells. An analysis of the two main LC subtypes AC and squamous cell carcinoma (SCC) revealed different expression patterns of miR-574-5p and the PGE2-synthesizing enzyme microsomal prostaglandin E synthase 1 (mPGES-1) in LC tissue sections and 3D tumor models. Further, it was demonstrated that PGE2 induces the secretion of sEV-derived miR-574-5p in LC cells via the activation of E prostanoid (EP) receptors 1/3. PGE2 inhibition significantly reduced miR-574-5p secretion, indicating that PGE2 levels specifically modulate miR-574-5p sorting into sEV. An analysis of tetraspanins, membrane proteins abundant in sEV, revealed different compositions between AC- and SCC-derived sEV. Finally, sEV-derived miR-574-5p proved its physiological function by activating Toll-like receptors (TLR) 7/8 in AC cells and decreasing intracellular miR-574-5p, mPGES-1, and PGE2 levels. Thus, AC cells revealed a novel function for miR-574-5p which depends on miR transmission via sEV and stands in contrast to its intracellular role. Together, intracellular- and sEV-derived miR-574-5p prevent uncontrolled upregulation of PGE2 via a negative feedback loop. The findings of this study demonstrate for the first time that an inflammatory mediator, here PGE2, induces the sorting of a miR into sEV and that sEV not only transfer miRs to recipient cells but can also change their mode of action

The multiple functions of miR-574-5p in the neuroblastoma tumor microenvironment

Neuroblastoma is the most common extracranial solid tumor in childhood and arises from neural crest cells of the developing sympathetic nervous system. Prostaglandin E₂ (PGE₂) has been identified as a key pro-inflammatory mediator of the tumor microenvironment (TME) that promotes neuroblastoma progression. We report that the interaction between the microRNA miR-574-5p and CUG-binding protein 1 (CUGBP1) induces the expression of microsomal prostaglandin E₂ synthase 1 (mPGES-1) in neuroblastoma cells, which contributes to PGE₂ biosynthesis. PGE₂ in turn specifically induces the sorting of miR-574-5p into small extracellular vesicles (sEV) in neuroblastoma cell lines. sEV are one of the major players in intercellular communication in the TME. We found that sEV-derived miR-574-5p has a paracrine function in neuroblastoma. It acts as a direct Toll-like receptor 7/8 (TLR7/8) ligand and induces α-smooth muscle actin (α-SMA) expression in fibroblasts, contributing to fibroblast differentiation. This is particularly noteworthy as it has an opposite function to that in the TME of lung carcinoma, another PGE₂ dependent tumor type. Here, sEV-derived miR-574-5p has an autokrine function that inhibits PGE₂ biosynthesis in lung cancer cells. We report that the tetraspanin composition on the surface of sEV is associated with the function of sEV-derived miR-574-5p. This suggests that the vesicles do not only transport miRs, but also appear to influence their mode of action

The multiple functions of miR-574-5p in the neuroblastoma tumor microenvironment

Neuroblastoma is the most common extracranial solid tumor in childhood and arises from neural crest cells of the developing sympathetic nervous system. Prostaglandin E₂ (PGE₂) has been identified as a key pro-inflammatory mediator of the tumor microenvironment (TME) that promotes neuroblastoma progression. We report that the interaction between the microRNA miR-574-5p and CUG-binding protein 1 (CUGBP1) induces the expression of microsomal prostaglandin E₂ synthase 1 (mPGES-1) in neuroblastoma cells, which contributes to PGE₂ biosynthesis. PGE₂ in turn specifically induces the sorting of miR-574-5p into small extracellular vesicles (sEV) in neuroblastoma cell lines. sEV are one of the major players in intercellular communication in the TME. We found that sEV-derived miR-574-5p has a paracrine function in neuroblastoma. It acts as a direct Toll-like receptor 7/8 (TLR7/8) ligand and induces α-smooth muscle actin (α-SMA) expression in fibroblasts, contributing to fibroblast differentiation. This is particularly noteworthy as it has an opposite function to that in the TME of lung carcinoma, another PGE₂ dependent tumor type. Here, sEV-derived miR-574-5p has an autokrine function that inhibits PGE₂ biosynthesis in lung cancer cells. We report that the tetraspanin composition on the surface of sEV is associated with the function of sEV-derived miR-574-5p. This suggests that the vesicles do not only transport miRs, but also appear to influence their mode of action

Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

The contraction and relaxation of the heart is controlled by stimulation of the beta 1-adrenoreceptor (AR) signaling cascade, which leads to activation of cAMP-dependent protein kinase (PKA) and subsequent cardiac protein phosphorylation. Phosphorylation is counteracted by the main cardiac protein phosphatases, PP2A and PP1. Both kinase and phosphatases are sensitive to intramolecular disulfide formation in their catalytic subunits that inhibits their activity. Additionally, intermolecular disulfide formation between PKA type I regulatory subunits (PKA-RI) has been described to enhance PKA's affinity for protein kinase A anchoring proteins, which alters its subcellular distribution. Nitroxyl donors have been shown to affect contractility and relaxation, but the mechanistic basis for this effect is unclear. The present study investigates the impact of several nitroxyl donors and the thiol-oxidizing agent diamide on cardiac myocyte protein phosphorylation and oxidation. Although all tested compounds equally induced intermolecular disulfide formation in PKA-RI, only 1-nitrosocyclohexalycetate (NCA) and diamide induced reproducible protein phosphorylation. Phosphorylation occurred independently of beta(1)-AR activation, but was abolished after pharmacological PKA inhibition and thus potentially attributable to increased PKA activity. NCA treatment of cardiac myocytes induced translocation of PKA and phosphatases to the myofilament compartment as shown by fractionation, immunofluorescence, and proximity ligation assays. Assessment of kinase and phosphatase activity within the myofilament fraction of cardiac myocytes after exposure to NCA revealed activation of PKA and inhibition of phosphatase activity thus explaining the increase in phosphorylation. The data suggest that the NCA-mediated effect on cardiac myocyte protein phosphorylation orchestrates alterations in the kinase/phosphatase balance