Determinación de nuevas funciones de la quinasa DYRK2 en respuesta a estrés genotóxico

Cancer is a complex disease enhanced by alterations in signaling pathways. In fact, DNA damage response (DDR) pathways play a key role in cancer development when they are deregulated. DDR pathways induce a cellular response that includes principally DNA repair pathways activation, cell cycle arrest, and cell death. Although there is a great knowledge of the signaling pathways that take place in DDR, further research is required to assess new molecular mechanisms for fully comprehend this complex scheme. In this sense, kinases like DYRK2, with an important role in DDR, are key to better understand and control DNA genomic damage situations. Thus, identification of new substrates for this kinase, as well as to elucidate novel pathways implicated in DDR would open a road to the development of new therapeutic strategies against cancer. In the present work we show NOTCH1 as a novel substrate for DYRK2. We describe for the first time a new regulation mechanism of the NOTCH1 signaling pathway mediated by this kinase. We demonstrate that DYRK2 phosphorylates Notch1-IC in response to DNA damage and facilitates its proteasomal degradation by FBXW7 ubiquitin ligase through a Thr2512 phosphorylation-dependent mechanism. We show that DNA damage-dependently triggered DYRK2 has a relevant effect on the viability, motility and invasion capacity of cancer cells expressing NOTCH1. In summary, we reveal a novel DRYK2-dependent mechanism of regulation for NOTCH1 which might help us to better understand its role in cancer biology. Besides, we attempted to overview of cellular circuits that are involved in ionizing radiation triggered DNA damage response in human dermal fibroblasts. To allow this, we employed three different approaches: RNA-seq and proteomic and phosphoproteomic analysis based on SILAC labelling. This is, to our knowledge, the first work that compare these 3 different functional approaches to this aim. The description of NOTCH1 as a new DYRK2 substrate, as well as the acquisition of solid data compilation that would permit a deeper understanding of the pathways that take place in response to ionizing radiation. This will provide new therapeutic opportunities for radiodermatitis prevention and treatment. Taken together, these data contribute to better understand the network of signaling pathways activationdeactivation under DNA damage conditions, opening a road to the development of new therapeutic strategies against cancer.El cáncer es una enfermedad compleja que se ve impulsada por alteraciones en rutas de señalización. De hecho, las rutas de respuesta al daño al ADN juegan un papel esencial en el desarrollo de cáncer cuando se encuentran desreguladas. Estas rutas inducen una respuesta celular que incluye principalmente la reparación del ADN, el arresto del ciclo celular y la muerte celular. A pesar de que existe un amplio conocimiento de las rutas de señalización de respuesta al daño al ADN, una mayor investigación es necesaria para detectar nuevos mecanismos moleculares que nos permitan comprender este complejo entramado de señales. En este sentido, proteínas como DYRK2 son esenciales para entender y controlar situaciones de daño al ADN. Por tanto, la identificación de nuevos sustratos para esta quinasa, así como de nuevas rutas de señalización implicadas en la respuesta al daño al ADN podrían ser herramientas muy útiles en el desarrollo de estrategias contra esta enfermedad. En este trabajo mostramos cómo NOTCH1 es un nuevo sustrato de DYRK2. Describimos por primera vez un nuevo mecanismo regulatorio de la ruta de señalización de Notch por medio de esta quinasa. Demostramos que DYRK2 fosforila a NOTCH1-IC en la Thr2512 en respuesta a daño al ADN y promueve su degradación proteosomal gracias a la ubiquitinación por parte de la ubiquitín-ligasa FBXW7. Además, mostramos que el efecto de DYRK2 en respuesta a estrés genotóxico tiene un efecto relevante en la viabilidad, motilidad y capacidad de invasión de células tumorales que expresan NOTCH1. En resumen, mostramos un nuevo mecanismo de regulación de NOTCH1 por parte de DYRK2 que podría ayudarnos a comprender mejor su papel en la biología del cáncer. Además, hemos tratado de obtener una visión general de los circuitos celulares implicados en la respuesta al daño al ADN generado por radiación ionizante en fibroblastos. Para ello hemos analizado 3 niveles ómicos distintos: transcriptómico (por RNA-seq), proteómico y fosfoproteómico (empleando SILAC). Este sería hasta ahora el primer trabajo que emplea estas 3 aproximaciones diferentes para este fin. En resumen, en este trabajo se describe NOTCH1 como un nuevo sustrato de DYRK2, así como la adquisición de datos sólidos que permiten una comprensión más profunda de las rutas que se activan en respuesta al daño al ADN. En conjunto, estos data contribuyen a una mejor comprensión de las redes de señalización que se activan y desactivan en respuesta al daño al ADN, abriendo una puerta al desarrollo de nuevas estrategias terapéuticas frente al cáncer

Phosphorylation-dependent regulation of the NOTCH1 intracellular domain by dual-specificity tyrosine-regulated kinase 2

NOTCH proteins constitute a receptor family with a widely conserved role in cell cycle, growing and development regulation. NOTCH1, the best characterised member of this family, regulates the expression of key genes in cell growth and angiogenesis, playing an essential role in cancer development. These observations provide a relevant rationale to propose the inhibition of the intracellular domain of NOTCH1 (Notch1-IC) as a strategy for treating various types of cancer. Notch1-IC stability is mainly controlled by post-translational modifications. FBXW7 ubiquitin E3 ligase-mediated degradation is considered one of the most relevant, being the previous phosphorylation at Thr-2512 residue required. In the present study, we describe for the first time a new regulation mechanism of the NOTCH1 signalling pathway mediated by DYRK2. We demonstrate that DYRK2 phosphorylates Notch1-IC in response to chemotherapeutic agents and facilitates its proteasomal degradation by FBXW7 ubiquitin ligase through a Thr-2512 phosphorylation-dependent mechanism. We show that DYRK2 regulation by chemotherapeutic agents has a relevant effect on the viability, motility and invasion capacity of cancer cells expressing NOTCH1. In summary, we reveal a novel mechanism of regulation for NOTCH1 which might help us to better understand its role in cancer biology

Hypothalamic miR-30 regulates puberty onset via repression of the puberty-suppressing factor, Mkrn3.

Mkrn3, the maternally imprinted gene encoding the makorin RING-finger protein-3, has recently emerged as putative pubertal repressor, as evidenced by central precocity caused by MKRN3 mutations in humans; yet, the molecular underpinnings of this key regulatory action remain largely unexplored. We report herein that the microRNA, miR-30, with three binding sites in a highly conserved region of its 3' UTR, operates as repressor of Mkrn3 to control pubertal onset. Hypothalamic miR-30b expression increased, while Mkrn3 mRNA and protein content decreased, during rat postnatal maturation. Neonatal estrogen exposure, causing pubertal alterations, enhanced hypothalamic Mkrn3 and suppressed miR-30b expression in female rats. Functional in vitro analyses demonstrated a strong repressive action of miR-30b on Mkrn3 3' UTR. Moreover, central infusion during the juvenile period of target site blockers, tailored to prevent miR-30 binding to Mkrn3 3' UTR, reversed the prepubertal down-regulation of hypothalamic Mkrn3 protein and delayed female puberty. Collectively, our data unveil a novel hypothalamic miRNA pathway, involving miR-30, with a prominent role in the control of puberty via Mkrn3 repression. These findings expand our current understanding of the molecular basis of puberty and its disease states

Hypothalamic miR-30 regulates puberty onset via repression of the puberty-suppressing factor, Mkrn3

Mkrn3, the maternally imprinted gene encoding the makorin RING-finger protein-3, has recently emerged as putative pubertal repressor, as evidenced by central precocity caused by MKRN3 mutations in humans; yet, the molecular underpinnings of this key regulatory action remain largely unexplored. We report herein that the microRNA, miR-30, with three binding sites in a highly conserved region of its 3 ' UTR, operates as repressor of Mkrn3 to control pubertal onset. Hypothalamic miR-30b expression increased, while Mkrn3 mRNA and protein content decreased, during rat postnatal maturation. Neonatal estrogen exposure, causing pubertal alterations, enhanced hypothalamic Mkrn3 and suppressed miR-30b expression in female rats. Functional in vitro analyses demonstrated a strong repressive action of miR-30b on Mkrn3 3 ' UTR. Moreover, central infusion during the juvenile period of target site blockers, tailored to prevent miR-30 binding to Mkrn3 3 ' UTR, reversed the prepubertal down-regulation of hypothalamic Mkrn3 protein and delayed female puberty. Collectively, our data unveil a novel hypothalamic miRNA pathway, involving miR-30, with a prominent role in the control of puberty via Mkrn3 repression. These findings expand our current understanding of the molecular basis of puberty and its disease states

Updating dual-specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2): molecular basis, functions and role in diseases.

Members of the dual-specificity tyrosine-regulated kinase (DYRKs) subfamily possess a distinctive capacity to phosphorylate tyrosine, serine, and threonine residues. Among the DYRK class II members, DYRK2 is considered a unique protein due to its role in disease. According to the post-transcriptional and post-translational modifications, DYRK2 expression greatly differs among human tissues. Regarding its mechanism of action, this kinase performs direct phosphorylation on its substrates or acts as a priming kinase, enabling subsequent substrate phosphorylation by GSK3β. Moreover, DYRK2 acts as a scaffold for the EDVP E3 ligase complex during the G2/M phase of cell cycle. DYRK2 functions such as cell survival, cell development, cell differentiation, proteasome regulation, and microtubules were studied in complete detail in this review. We have also gathered available information from different bioinformatic resources to show DYRK2 interactome, normal and tumoral tissue expression, and recurrent cancer mutations. Then, here we present an innovative approach to clarify DYRK2 functionality and importance. DYRK2 roles in diseases have been studied in detail, highlighting this kinase as a key protein in cancer development. First, DYRK2 regulation of c-Jun, c-Myc, Rpt3, TERT, and katanin p60 reveals the implication of this kinase in cell-cycle-mediated cancer development. Additionally, depletion of this kinase correlated with reduced apoptosis, with consequences on cancer patient response to chemotherapy. Other functions like cancer stem cell formation and epithelial-mesenchymal transition regulation are also controlled by DYRK2. Furthermore, the pharmacological modulation of this protein by different inhibitors (harmine, curcumine, LDN192960, and ID-8) has enabled to clarify DYRK2 functionality

Stem/progenitor cells in normal physiology and disease of the pancreas

Though embryonic pancreas progenitors are well characterised, the existence of stem/progenitor cells in the postnatal mammalian pancreas has been long debated, mainly due to contradicting results on regeneration after injury or disease in mice. Despite these controversies, sequencing advancements combined with lineage tracing and organoid technologies indicate that homeostatic and trigger-induced regenerative responses in mice could occur. The presence of putative progenitor cells in the adult pancreas has been proposed during homeostasis and upon different stress challenges such as inflammation, tissue damage and oncogenic stress. More recently, single cell transcriptomics has revealed a remarkable heterogeneity in all pancreas cell types, with some cells showing the signature of potential progenitors. In this review we provide an overview on embryonic and putative adult pancreas progenitors in homeostasis and disease, with special emphasis on in vitro culture systems and scRNA-seq technology as tools to address the progenitor nature of different pancreatic cells

FBXW7 tumor suppressor regulation by dualspecificity tyrosine-regulated kinase 2

FBXW7 is a member of the F-box protein family, which functions as the substrate recognition component of the SCF E3 ubiquitin ligase. FBXW7 is a main tumor suppressor due to its ability to control proteasome-mediated degradation of several oncoproteins such as c-Jun, c-Myc, Cyclin E1, mTOR, and Notch1-IC. FBXW7 inactivation in human cancers results from a somatic mutation or downregulation of its protein levels. This work describes a novel regulatory mechanism for FBXW7 dependent on the serine/threonine protein kinase DYRK2. We show that DYRK2 interacts with and phosphorylates FBXW7 resulting in its proteasome-mediated degradation. DYRK2-dependent FBXW7 destabilization is independent of its ubiquitin ligase activity. The functional analysis demonstrates the existence of DYRK2-dependent regulatory mechanisms for key FBXW7 substrates. Finally, we provide evidence indicating that DYRK2-dependent regulation of FBXW7 protein accumulation contributes to cytotoxic effects in response to chemotherapy agents such as Doxorubicin or Paclitaxel in colorectal cancer cell lines and to BET inhibitors in T-cell acute lymphoblastic leukemia cell lines. Altogether, this work reveals a new regulatory axis, DYRK2/FBXW7, which provides an understanding of the role of these two proteins in tumor progression and DNA damage responses.This work was funded by the Spanish Ministerio de Ciencia e Innovación (MICINN, PID2021-124314OB-I00 to MAC and PID2019-107185GB-I00 to SdlL), Junta de Andalucía-Consejería de Conocimiento, Investigación y Universidad (P20_00470 to MAC) and University of Cordoba (1380920-R to MAC) grants. ACS and LCS were supported by an FPU fellowship (FPU18/ 00845 and FPU20/02699, respectively) from the Spanish Ministerio de Educación y Formación Profesional

Tetrahydrocannabinolic acid A (THCA-A) reduces adiposity and prevents metabolic disease caused by diet-induced obesity.

Medicinal cannabis has remarkable therapeutic potential, but its clinical use is limited by the psychotropic activity of Δ9-tetrahydrocannabinol (Δ9-THC). However, the biological profile of the carboxylated, non-narcotic native precursor of Δ9-THC, the Δ9-THC acid A (Δ9-THCA-A), remains largely unexplored. Here we present evidence that Δ9-THCA-A is a partial and selective PPARγ modulator, endowed with lower adipogenic activity than the full PPARγ agonist rosiglitazone (RGZ) and enhanced osteoblastogenic effects in hMSC. Docking and in vitro functional assays indicated that Δ9-THCA-A binds to and activates PPARγ by acting at both the canonical and the alternative sites of the ligand-binding domain. Transcriptomic signatures in iWAT from mice treated with Δ9-THCA-A confirmed its mode of action through PPARγ. Administration of Δ9-THCA-A in a mouse model of HFD-induced obesity significantly reduced fat mass and body weight gain, markedly ameliorating glucose intolerance and insulin resistance, and largely preventing liver steatosis, adipogenesis and macrophage infiltration in fat tissues. Additionally, immunohistochemistry, transcriptomic, and plasma biomarker analyses showed that treatment with Δ9-THCA-A caused browning of iWAT and displayed potent anti-inflammatory actions in HFD mice. Our data validate the potential of Δ9-THCA-A as a low adipogenic PPARγ agonist, capable of substantially improving the symptoms of obesity-associated metabolic syndrome and inflammation

A novel CDC25A/DYRK2 regulatory switch modulates cell cycle and survival

Data de publicació electrònica: 06-08-2021The cell division cycle 25A (CDC25A) phosphatase is a key regulator of cell cycle progression that acts on the phosphorylation status of Cyclin-Cyclin-dependent kinase complexes, with an emergent role in the DNA damage response and cell survival control. The regulation of CDC25A activity and its protein level is essential to control the cell cycle and maintain genomic integrity. Here we describe a novel ubiquitin/proteasome-mediated pathway negatively regulating CDC25A stability, dependent on its phosphorylation by the serine/threonine kinase DYRK2. DYRK2 phosphorylates CDC25A on at least 7 residues, resulting in its degradation independent of the known CDC25A E3 ubiquitin ligases. CDC25A in turn is able to control the phosphorylation of DYRK2 at several residues outside from its activation loop, thus affecting DYRK2 localization and activity. An inverse correlation between DYRK2 and CDC25A protein amounts was observed during cell cycle progression and in response to DNA damage, with CDC25A accumulation responding to the manipulation of DYRK2 levels or activity in either physiological scenario. Functional data show that the pro-survival activity of CDC25A and the pro-apoptotic activity of DYRK2 could be partly explained by the mutual regulation between both proteins. Moreover, DYRK2 modulation of CDC25A expression and/or activity contributes to the DYRK2 role in cell cycle regulation. Altogether, we provide evidence suggesting that DYRK2 and CDC25A mutually control their activity and stability by a feedback regulatory loop, with a relevant effect on the genotoxic stress pathway, apoptosis, and cell cycle regulation.This work was funded by Ministerio de Ciencia e Innovación (SAF2016-75228-R to MAC and BFU2016-76141-P to SdlL) and by Cancer Research UK (C52419/A22869) to LdlV. MLC and ACS were supported by a FPU fellowship (FPU13/03393 and FPU18/00845, respectively) from the Ministerio de Educación y Formación Profesional