Uridine 5′-triphosphate promotes in vitro Schwannoma cell migration through matrix metalloproteinase-2 activation

In response to peripheral nerve injury, Schwann cells adopt a migratory phenotype and modify the extracellular matrix to make it permissive for cell migration and axonal re-growth. Uridine 5′-triphosphate (UTP) and other nucleotides are released during nerve injury and activate purinergic receptors expressed on the Schwann cell surface, but little is known about the involvement of purine signalling in wound healing. We studied the effect of UTP on Schwannoma cell migration and wound closure and the intracellular signaling pathways involved. We found that UTP treatment induced Schwannoma cell migration through activation of P2Y2 receptors and through the increase of extracellular matrix metalloproteinase-2 (MMP-2) activation and expression. Knockdown P2Y2 receptor or MMP-2 expression greatly reduced wound closure and MMP-2 activation induced by UTP. MMP-2 activation evoked by injury or UTP was also mediated by phosphorylation of all 3 major mitogen-activated protein kinases (MAPKs): JNK, ERK1/2, and p38. Inhibition of these MAPK pathways decreased both MMP-2 activation and cell migration. Interestingly, MAPK phosphorylation evoked by UTP exhibited a biphasic pattern, with an early transient phosphorylation 5 min after treatment, and a late and sustained phosphorylation that appeared at 6 h and lasted up to 24 h. Inhibition of MMP-2 activity selectively blocked the late, but not the transient, phase of MAPK activation. These results suggest that MMP-2 activation and late MAPK phosphorylation are part of a positive feedback mechanism to maintain the migratory phenotype for wound healing. In conclusion, our findings show that treatment with UTP stimulates in vitro Schwannoma cell migration and wound repair through a MMP-2-dependent mechanism via P2Y2 receptors and MAPK pathway activation. © 2014 Lamarca et al.This research was supported by an unrestricted research grant from Ferrer S.A. (Barcelona, Spain) and by grant SAF2011-23550 from Ministerio de Economia y Competitividad of SpainPeer Reviewe

Estudio del fármaco CMP Forte y del nucleótido UTP en células de Schwann

Las neuropatías periféricas representan una de las mayores complicaciones del SNP. Pocos son los fármacos efectivos para su tratamiento y la mayoría se basan en el tratamiento del dolor. Núcleo CMP Forte® es un fármaco compuesto por los nucleótidos (UMP, UDP, UTP y CMP) que se prescribe a pacientes con neuropatías periféricas de diverso origen; sin embargo, se desconoce cuál es su mecanismo de acción. Los resultados de la presente tesis demuestran que la presencia de nucleótidos extracelulares en las células de Schwann provocan una reestructuración del citoesqueleto de actina y aumentos en la adhesión (N cadherina) de forma dependiente a la activación de P2YR y de tres MAPK (JNK, P38 Y ERK1/2). Como conclusión podemos decir que los efectos a través de nuc!eótidos extracelulares podrían beneficiar los procesos de migración y de adhesión en presencia de daño axonal, favoreciendo los primeros contactos intercelulares entre células de Schwann y axones antes de remielinizar y regenerar el daño provocado en el SNP.Peripheral neuropathies represent one of the most common pathology in the PNS. Currently, there are no efficient treatments for peripheral neuropathies, and the drugs administrated are based in pain treatment. Núcleo CMP Forte® is a drug composed by the nucleotides (UMP, UDP, UTP y CMP) and is prescribed to patients with PNS neuropathies. Despite Núcleo CMP Forte® administration leads to improvement in the patients there is no information about its mechanism of action. Our results demonstrated that extracellular nucleotides change actin organization and cellular adhesion (N cadherin) in Schwann cells. They depend on UTP-induced P2YR and MAPK (ERK1/2, JNK and P38) activation. To sum up, extracellular nucleotides effects could be beneficial in migration and adhesion processes promoting the first contacts between axons and Schwann cells and improving the regeneration in the presence oxonal damage in PNS

Targeting miRNA-551b, a "Stemness"-like microRNA, to Eradicate AML (Stem) Cells

Despite high complete remission (CR) rates achieved after chemotherapy, only 30-40% of patients with Acute Myeloid Leukemia (AML) survive five years after diagnosis. The main cause of this treatment failure is insufficient eradication of a subpopulation of chemotherapy-resistant leukemia cells with stem cell properties, named "leukemic stem cells" (LSCs). LSCs use a variety of mechanisms to resist chemotherapy and targeting them is one of the major challenges in AML treatment. Since miRNAs can target multiple genes/pathways simultaneously, their modulation (downregulation or upregulation) may have great potential for the successful elimination of therapy-resistant leukemic (stem) cells (Martiañez Canales et al. Cancers 2017). Here, we show that miRNA-551b, previously identified by us as a stem cell-like miRNA, can be a potential novel target to specifically eradicate AML stem-like cells.Aiming at identification of miRNA-based therapy to specifically eradicate LSCs, while sparing normal Hematopoietic Stem Cells (HSCs), we determined expression of miRNAs in normal HSCs, Leukemic Stem Cells (LSCs) and leukemic progenitors (LP) all derived from the same AML patient's bone marrow. Using this approach, we identified miRNA-551b as being highly expressed in normal HSCs residing both in healthy and AML bone marrows. In AML, high expression of miR551b demonstrated to be associated with an adverse prognosis. Moreover, miRNA-551b was highly expressed in immature AML cases and its expression in a cohort of patients coincided with the expression of stem cell genes (De Leeuw et al. Leukemia 2016).To further elucidate the link between miRNA-551b and AML "stemness" and to test whether downregulation of miRNA-551b affects the survival of AML (stem/progenitor) cells, proliferation and the balance between differentiation and "stemness", we reduced miRNA-551b expression, either by lentiviral transduction of antagomirs or by adding locked nucleotide acid (LNA)-oligonucleotides to AML cell lines and primary AML cells. Downregulation of miRNA-551b in the stem cell-like AML cell line KG1a led to inhibition of cell growth in vitro, which was due to inhibition of proliferation rather than induction of apoptosis. KG1a tumor growth in an in vivo mouse model was also reduced when miRNA-551b was downregulated. In primary AML, miRNA-551b knockdown resulted in a significant decrease in the survival of leukemic progenitors and LSCs, while hematopoietic stem cells (HSCs) and normal progenitors from healthy bone marrows were not affected. These results suggest that a therapeutic approach inhibiting miRNA-551b expression might specifically eradicate leukemic progenitors and LSCs from primary AML, while sparing HSCs. We are currently studying miRNA-551b targets which can be responsible for this specific LSCs elimination.In conclusion, our results suggest that inhibition of miRNA-551b could be a promising approach to eliminate stem cell-like AML cells, thereby decreasing relapse rates and improving AML treatment outcome.Disclosures Ossenkoppele: Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Research Funding; Roche: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Johnson & Johnson: Consultancy, Honoraria, Research Funding; Genmab: Research Funding.↵* Asterisk with author names denotes non-ASH members