Unconventional cell cycles in polyploidization: the megakaryocyte paradigm

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 01-07-201

Proliferative advantage of specific aneuploid cells drives evolution of tumor karyotypes

Most tumors have abnormal karyotypes, which arise from mistakes during mitotic division of healthy euploid cells and evolve through numerous complex mechanisms. In a recent mouse model with increased chromosome missegregation, chromosome gains dominate over losses both in pretumor and tumor tissues, whereas T-cell lymphomas are characterized by gains of chromosomes 14 and 15. However, the quantitative understanding of clonal selection leading to tumor karyotype evolution remains unknown. Here we show, by introducing a mathematical model based on a concept of a macro-karyotype, that tumor karyotypes can be explained by proliferation-driven evolution of aneuploid cells. In pretumor cells, increased apoptosis and slower proliferation of cells with monosomies lead to predominant chromosome gains over losses. Tumor karyotypes with gain of one chromosome can be explained by karyotype-dependent proliferation, whereas, for those with two chromosomes, an interplay with karyotype-dependent apoptosis is an additional possible pathway. Thus, evolution of tumor-specific karyotypes requires proliferative advantage of specific aneuploid karyotypes

Thrombocytopenia-associated mutations in Ser/Thr kinase MASTL deregulate actin cytoskeleton dynamics in platelets

MASTL, a Ser/Thr kinase that inhibits PP2A-B55 complexes during mitosis, is mutated in autosomal dominant thrombocytopenia. However, the connections between the cell cycle machinery and this human disease remain unexplored. We report here that, whereas Mastl ablation in megakaryocytes prevented proper maturation of these cells, mice carrying the thrombocytopenia-associated mutation developed thrombocytopenia as a consequence of aberrant activation and survival of platelets. Activation of mutant platelets was characterized by hyper-stabilized pseudopods mimicking the effect of PP2A inhibition and actin polymerization defects. These aberrations were accompanied by abnormal hyper-phosphorylation of multiple components of the actin cytoskeleton and were rescued both in vitro and in vivo by inhibiting upstream kinases such as PKA, PKC, or AMPK. These data reveal an unexpected role of Mastl in actin cytoskeleton dynamics in postmitotic cells, and suggest that the thrombocytopenia-associated mutation in MASTL is a pathogenic dominant mutation that mimics decreased PP2A activity resulting in altered phosphorylation of cytoskeletal regulatory pathways.We thank Peter Storz (Mayo Clinic; Jacksonville, FL) for sharin g reagents and Sheila Rueda for her support with the management of the mouse colony. B.H. and R.S.-M. were supported by the Juan de la Cierva Programme from the Spanish M inistry of Economy and Competitiveness (MINECO). M.T. was supported by Foundation La Caixa. A.E.B. was supported by the Programa de Empleo Juvenil, Comunidad de M adrid. M.A.-F. received a young investigator g rant from MINECO (SAF2014-60442- JIN; co-financed by FEDER funds). P.G.dF. was supported by Fundació la Marató de TV3 (project 080121 and project 20153031). J.M. was supported by the Ramon y Cajal programme (MINECO; RYC-2012-10651). M.M. lab. is supported by grants from the MINE CO (SAF2015- 69920-R), Programa iLUNG from the Comunidad de Madrid (B2017/BM D-3884), and Worldwide Cancer Research (15-0278). CNIO is a Severo Ochoa Cen ter of Excellence (MINECO awards SEV-2015-0510)S

Transient exposure to miR-203 expands the differentiation capacity of pluripotent stem cells

Full differentiation potential along with self‐renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro . We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already‐established murine and human PSCs. Short exposure to miR‐203 in PSCs (mi PSCs) induces a transient expression of 2C markers that later results in expanded differentiation potency to multiple lineages, as well as improved efficiency in tetraploid complementation and human–mouse interspecies chimerism assays. Mechanistically, these effects are at least partially mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasure of DNA methylation. These data support the use of transient exposure to miR‐203 as a versatile method to reset the epigenetic memory in PSCs, and improve their effectiveness in regenerative medicine

p38γ is essential for cell cycle progression and liver tumorigenesis

The cell cycle is a tightly regulated process that is controlled by the conserved cyclin-dependent kinase (CDK)–cyclin protein complex1. However, control of the G0-to-G1 transition is not completely understood. Here we demonstrate that p38 MAPK gamma (p38γ) acts as a CDK-like kinase and thus cooperates with CDKs, regulating entry into the cell cycle. p38γ shares high sequence homology, inhibition sensitivity and substrate specificity with CDK family members. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of retinoblastoma tumour suppressor protein at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor pirfenidone protects against the chemically induced formation of liver tumours. Furthermore, biopsies of human hepatocellular carcinoma show high expression of p38γ, suggesting that p38γ could be a therapeutic target in the treatment of this disease

Regulation of macrophage activation and septic shock susceptibility via p21(WAF1/CIP1)

p21 is a cell-cycle inhibitor that is also known to suppress autoimmunity. Here, we provide evidence of a novel role for p21 as an inhibitor of macrophage activation. LPS stimulation of p21-deficient peritoneal macrophages induced increased activation compared with controls, with elevated production of proinflammatory mediators such as TNF-α and IL-1β. The enhanced activity of LPS-stimulated p21-deficient macrophages correlated with increased activity of the transcription factor NF-κB. LPS stimulation of p21-deficient macrophages led to increased IκBα kinase activity, and increased IκBα phosphorylation and degradation, resulting in elevated NF-κB activity. The effect of p21 in macrophage activation was independent of its cell-cycle inhibitory role. p21−/− mice showed greater sensitivity to LPS-induced septic shock than did WT mice, indicating that p21 contributes to maintenance of a balanced response to inflammatory stimuli and suggesting biological significance for the role of p21 in macrophage activation. Our findings project a role for p21 in the control of NF-κB-associated inflammation, and suggest that therapeutic modulation of p21 expression could be beneficial in inflammation-associated diseases.Ministerio de Economía y CompetitividadDepto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

Activation of the endomitotic spindle assembly checkpoint and thrombocytopenia in Plk1-deficient mice.

Polyploidization in megakaryocytes is achieved by endomitosis, a specialized cell cycle in which DNA replication is followed by aberrant mitosis. Typical mitotic regulators such as Aurora kinases or Cdk1 are dispensable for megakaryocyte maturation, and inhibition of mitotic kinases may in fact promote megakaryocyte maturation. However, we show here that Polo-like kinase 1 (Plk1) is required for endomitosis, and ablation of the Plk1 gene in megakaryocytes results in defective polyploidization accompanied by mitotic arrest and cell death. Lack of Plk1 results in defective centrosome maturation and aberrant spindle pole formation, thus impairing the formation of multiple poles typically found in megakaryocytes. In these conditions, megakaryocytes arrest for a long time in mitosis and frequently die. Mitotic arrest in wild-type megakaryocytes treated with Plk1 inhibitors or Plk1-null cells is triggered by the spindle assembly checkpoint (SAC), and can be rescued in the presence of SAC inhibitors. These data suggest that, despite the dispensability of proper chromosome segregation in megakaryocytes, an endomitotic SAC is activated in these cells upon Plk1 inhibition. SAC activation results in defective maturation of megakaryocytes and cell death, thus raising a note of caution in the use of Plk1 inhibitors in therapeutic strategies based on polyploidization regulators.This work was supported by a fellowship from the Foundation La Caixa (M.T.), and the Cell Division and Cancer group of the CNIO was funded by the Ministry of Economy and Competitiveness (SAF2012-38215), Consolider-Ingenio 2010 Programme (SAF2014-57791-REDC), Red Tematica CellSYS (BFU2014-52125-REDT), Comunidad de Madrid (OncoCycle Programme, S2010/BMD-2470), Worldwide Cancer Research (WCR #15-0278), and the MitoSys project (HEALTH-F5-2010-241548, European Union Seventh Framework Programme).S

Functional reprogramming of polyploidization in megakaryocytes.

Polyploidization is a natural process that frequently accompanies differentiation; its deregulation is linked to genomic instability and cancer. Despite its relevance, why cells select different polyploidization mechanisms is unknown. Here we report a systematic genetic analysis of endomitosis, a process in which megakaryocytes become polyploid by entering mitosis but aborting anaphase. Whereas ablation of the APC/C cofactor Cdc20 results in mitotic arrest and severe thrombocytopenia, lack of the kinases Aurora-B, Cdk1, or Cdk2 does not affect megakaryocyte polyploidization or platelet levels. Ablation of Cdk1 forces a switch to endocycles without mitosis, whereas polyploidization in the absence of Cdk1 and Cdk2 occurs in the presence of aberrant re-replication events. Importantly, ablation of these kinases rescues the defects in Cdc20 null megakaryocytes. These findings suggest that endomitosis can be functionally replaced by alternative polyploidization mechanisms in vivo and provide the cellular basis for therapeutic approaches aimed to discriminate mitotic and polyploid cells.S

Activation of the endomitotic spindle assembly checkpoint and thrombocytopenia in Plk1-deficient mice

Polyploidization in megakaryocytes is achieved by endomitosis, a specialized cell cycle in which DNA replication is followed by aberrant mitosis. Typical mitotic regulators such as Aurora kinases or Cdk1 are dispensable for megakaryocyte maturation, and inhibition of mitotic kinases may in fact promote megakaryocyte maturation. However, we show here that Polo-like kinase 1 (Plk1) is required for endomitosis, and ablation of the Plk1 gene in megakaryocytes results in defective polyploidization accompanied by mitotic arrest and cell death. Lack of Plk1 results in defective centrosome maturation and aberrant spindle pole formation, thus impairing the formation of multiple poles typically found in megakaryocytes. In these conditions, megakaryocytes arrest for a long time in mitosis and frequently die. Mitotic arrest in wild-type megakaryocytes treated with Plk1 inhibitors or Plk1-null cells is triggered by the spindle assembly checkpoint (SAC), and can be rescued in the presence of SAC inhibitors. These data suggest that, despite the dispensability of proper chromosome segregation in megakaryocytes, an endomitotic SAC is activated in these cells upon Plk1 inhibition. SAC activation results in defective maturation of megakaryocytes and cell death, thus raising a note of caution in the use of Plk1 inhibitors in therapeutic strategies based on polyploidization regulators.Depto. de Bioquímica y Biología MolecularFac. de Ciencias BiológicasTRUEpu

miR-203 imposes an intrinsic barrier during cellular reprogramming by targeting NFATC2

Cellular reprogramming from somatic to pluripotent cells is the basis for multiple applications aimed to replace damaged tissues in regenerative medicine. However, this process is limited by intrinsic barriers that are induced in response to reprogramming factors. In this manuscript we report that miR-203, a microRNA with multiple functions in differentiation and tumor suppression, acts as an endogenous barrier to reprogramming. Genetic ablation of miR-203 results in enhanced reprogramming whereas its expression prevents the formation of pluripotent cells both in vitro and in vivo. Mechanistically, this effect correlates with the direct repression of NFATC2, a transcription factor involved in the early phases of reprogramming. Inhibition of NFATC2 mimics miR-203 effects whereas NFATC2 overexpression rescues inducible cell pluripotency in miR-203- overexpressing cultures. These data suggest that miR-203 repression may favor the efficiency of reprogramming in a variety of cellular models.Depto. de Bioquímica y Biología MolecularFac. de Ciencias BiológicasFALSEunpu