Glycogen Synthase Kinase 3 (GSK-3) involvement in regulation of mouse embryonic stem cell fate

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Controlling embryonic stem cell proliferation and pluripotency: the role of PI3K-and GSK-3- dependent signalling

Abstract ESCs (embryonic stem cells) are derived from the inner cell mass of pre-implantation embryos and are pluripotent, meaning they can differentiate into all of the cells that make up the adult organism. This property of pluripotency makes ESCs attractive as a model system for studying early development and for the generation of specific cell types for use in regenerative medicine and drug screening. In order to harness their potential, the molecular mechanisms regulating ESC pluripotency, proliferation and differentiation (i.e. cell fate) need to be understood so that pluripotency can be maintained during expansion, while differentiation to specific lineages can be induced accurately when required. The present review focuses on the potential roles that PI3K (phosphoinositide 3-kinase) and GSK-3 (glycogen synthase kinase 3)-dependent signalling play in the co-ordination and integration of mouse ESC pluripotency and proliferation and contrast this with our understanding of their functions in human ESCs. Control of ESC (embryonic stem cell) fate: an overview ESCs are derived from early pre-implantation embryos and, when cultured appropriately, can be maintained in a proliferative, self-renewing and pluripotent state almost indefinitely. Pluripotency is the ability to differentiate into all of the cells found in an adult organism, while self-renewal describes the generation of a daughter stem cell from its mother. In the case of ESCs, self-renewal occurs symmetrically, such that when an undifferentiated ESC divides and pluripotency is maintained, both its progeny will be undifferentiated Over the last 5-10 years, our understanding of the molecular components involved in maintaining pluripotency of mESCs (mouse ESCs) has increased dramatically, from a simple 'prelude' where STAT3 (signal transducer and activator of transcription 3) activation by LIF (leukaemia Key words: cell cycle, embryonic stem cell, glycogen synthase kinase 3 (GSK-3), phosphoinositide 3-kinase (PI3K), pluripotency, proliferation, self-renewal. Abbreviations used: CDK, cyclin-dependent kinase; ESC, embryonic stem cell; Esrrb, oestrogenrelated receptor β; GSK-3, glycogen synthase kinase-3; hESC, human ESC; LIF, leukaemia inhibitory factor; MEK, mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase; mESC, mouse ESC; miRNA, microRNA; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; siRNA, short interfering RNA. 1 To whom correspondence should be addressed (email [email protected]). inhibitory factor) was all that seemed necessary, to a complex 'symphony' where extrinsic factors, intracellular signals, transcription factors, epigenetic regulators and miRNAs (microRNAs) have all been implicated The ESC cell cycle mESCs proliferate rapidly in culture and display unique cell-cycle kinetics, distinct from those of somatic cells, dividing approximately every 11-16 h and exhibiting a shortened G 1 -phase INK4a [13] and neither do mESCs arrest following DNA damag

The miR-17-92 cluster counteracts quiescence and chemoresistance in a distinct subpopulation of pancreatic cancer stem cells

Objective Cancer stem cells (CSCs) represent the root of many solid cancers including pancreatic ductal adenocarcinoma, are highly chemoresistant and represent the cellular source for disease relapse. However the mechanisms involved in these processes still need to be fully elucidated. Understanding the mechanisms implicated in chemoresistance and metastasis of pancreatic cancer is critical to improving patient outcomes. Design Micro-RNA (miRNA) expression analyses were performed to identify functionally defining epigenetic signatures in pancreatic CSC-enriched sphere-derived cells and gemcitabine-resistant pancreatic CSCs. Results We found the miR-17-92 cluster to be downregulated in chemoresistant CSCs versus non-CSCs and demonstrate its crucial relevance for CSC biology. In particular, overexpression of miR-17-92 reduced CSC self-renewal capacity, in vivo tumourigenicity and chemoresistance by targeting multiple NODAL/ACTIVIN/TGF-beta 1 signalling cascade members as well as directly inhibiting the downstream targets p21, p57 and TBX3. Overexpression of miR-17-92 translated into increased CSC proliferation and their eventual exhaustion via downregulation of p21 and p57. Finally, the translational impact of our findings could be confirmed in preclinical models for pancreatic cancer. Conclusions Our findings therefore identify the miR-17-92 cluster as a functionally determining family of miRNAs in CSCs, and highlight the putative potential of developing modulators of this cluster to overcome drug resistance in pancreatic CSCs.CH: ERC Advanced Investigator Grant (Pa-CSC 233460), European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement No 256974 (EPC-TM-NET) and No 602783 (CAM-PaC), the Subdireccion General de Evaluacion y Fomento de la Investigacion, Fondo de Investigacion Sanitaria (PS09/02129 \& PI12/02643), and the Programa Nacional de Internacionalizacion de la I+D, Subprogramma: FCCI 2009 (PLE2009-0105; Ministerio de Economia y Competitividad, Spain). MC: La Caixa Predoctoral Fellowship.S

Microenvironmental hCAP-18/LL-37 promotes pancreatic ductal adenocarcinoma by activating its cancer stem cell compartment

This is the peer reviewed version of the following article: Microenvironmental hCAP-18/LL-37 promotes pancreatic ductal adenocarcinoma by activating its cancer stem cell compartment. Gut 64.12 (2015): 1921-1935 and which has been published in final form at http://dx.doi.org/10.1136/gutjnl-2014-308935OBJECTIVES: The tumour stroma/microenvironment not only provides structural support for tumour development, but more importantly it provides cues to cancer stem cells (CSCs) that regulate their self-renewal and metastatic potential. This is certainly true for pancreatic ductal adenocarcinomas (PDAC), where tumour-associated fibroblasts, pancreatic stellate cells and immune cells create an abundant paracrine niche for CSCs via microenvironment-secreted factors. Thus understanding the role that tumour stroma cells play in PDAC development and CSC biology is of utmost importance. DESIGN: Microarray analyses, tumour microarray immunohistochemical assays, in vitro co-culture experiments, recombinant protein treatment approaches and in vivo intervention studies were performed to understand the role that the immunomodulatory cationic antimicrobial peptide 18/LL-37 (hCAP-18/LL-37) plays in PDAC biology. RESULTS: We found that hCAP-18/LL-37 was strongly expressed in the stroma of advanced primary and secondary PDAC tumours and is secreted by immune cells of the stroma (eg, tumour-associated macrophages) in response to tumour growth factor-β1 and particularly CSC-secreted Nodal/ActivinA. Treatment of pancreatic CSCs with recombinant LL-37 increased pluripotency-associated gene expression, self-renewal, invasion and tumourigenicity via formyl peptide receptor 2 (FPR2)- and P2X purinoceptor 7 receptor (P2X7R)-dependent mechanisms, which could be reversed by inhibiting these receptors. Importantly, in a genetically engineered mouse model of K-Ras-driven pancreatic tumourigenesis, we also showed that tumour formation was inhibited by either reconstituting these mice with bone marrow from cathelicidin-related antimicrobial peptide (ie, murine homologue of hCAP-18/LL-37) knockout mice or by pharmacologically inhibiting FPR2 and P2X7R. CONCLUSIONS: Thus, hCAP-18/LL-37 represents a previously unrecognised PDAC microenvironment factor that plays a critical role in pancreatic CSC-mediated tumourigenesis.CH: ERC Advanced Investigator Grant (Pa-CSC 233460), European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 256974 (EPC-TM-NET) and n° 602783 (CAM-PaC), the Subdirección General de Evaluación y Fomento de la Investigación, Fondo de Investigación Sanitaria (PS09/02129 & PI12/02643) and the Programa Nacional de Internacionalización de la I+D, Subprogramma: FCCI 2009 (PLE2009-0105; both Ministerio de Economía y Competitividad (es), Spain), BSJr: Rámon y Cajal Merit Award from the Ministerio de Economía y Competitividad, Spain and Clinic and Laboratory Integration Program (CLIP) grant from the Cancer Research Institute, NY, NY. MC: La Caixa Predoctoral Fellowshi

Differential Coupling of Self-Renewal Signaling Pathways in Murine Induced Pluripotent Stem Cells

The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs), exhibiting properties similar to those of embryonic stem cells (ESCs), has attracted much attention, with many studies focused on improving efficiency of derivation and unraveling the mechanisms of reprogramming. Despite this widespread interest, our knowledge of the molecular signaling pathways that are active in iPSCs and that play a role in controlling their fate have not been studied in detail. To address this shortfall, we have characterized the influence of different signals on the behavior of a model mouse iPSC line. We demonstrate significant responses of this iPSC line to the presence of serum, which leads to profoundly enhanced proliferation and, depending on the medium used, a reduction in the capacity of the iPSCs to self-renew. Surprisingly, this iPSC line was less sensitive to withdrawal of LIF compared to ESCs, exemplified by maintenance of expression of a Nanog-GFP reporter and enhanced self-renewal in the absence of LIF. While inhibition of phosphoinositide-3 kinase (PI3K) signaling decreased iPSC self-renewal, inhibition of Gsk-3 promoted it, even in the absence of LIF. High passages of this iPSC line displayed altered characteristics, including genetic instability and a reduced ability to self-renew. However, this second feature could be restored upon inhibition of Gsk-3. Collectively, our data suggest modulation of Gsk-3 activity plays a key role in the control of iPSC fate. We propose that more careful consideration should be given to characterization of the molecular pathways that control the fate of different iPSC lines, since perturbations from those observed in naïve pluripotent ESCs could render iPSCs and their derivatives susceptible to aberrant and potentially undesirable behaviors

Effect of perturbation of key self-renewal signaling pathways in ESCs and iPSCs.

<p>ESCs and iPSCs were seeded in KO SR plus or minus LIF as indicated. After 24 hours 2 µM 1 m and 10% (v/v) Hyclone serum (HY) alone or together (<b>A</b>), 2 µM 1 m (<b>B</b>), 5 µM LY294002 and 10% (v/v) HY (<b>C</b>) or DMSO 1∶10000 (in all controls), were added. After a further 24 hours proteins were extracted and immunoblotting performed as indicated. Blots were stripped and re-probed with Gapdh, pan Akt or Shp2 antibodies to assess loading.</p

Expression and phosphorylation status of key signaling intermediates in ESCs and iPSCs.

<p><b>A</b> and <b>B</b> ESCs or iPSCs were seeded 8000 cells/cm² in KO SR plus LIF. After 24 hours, 10% (v/v) Hyclone serum (HY) was added where indicated (<b>A</b>), or cells were washed 3 times with PBS before LIF-containing or LIF-free KO SR was added to the cultures as indicated (<b>B</b>). After a further 24 hours incubation, protein extracts were prepared, separated through 10% acrylamide gels using SDS-PAGE and immunoblotted using the antibodies indicated. Blots in <b>A</b> and <b>B</b>(i) were all generated using cell extracts from one replicate and those in <b>B</b>(ii) from an independent experimental replicate. <b>C</b> ESCs or iPSCs were seeded at 8500 cells/cm² in GMEM 10% (v/v) Hyclone serum (HY) plus LIF, then 24 h later washed and deprived of LIF and serum for 4 h. 5000 U/ml of LIF or 10% (v/v) HY were added and proteins extracted following 0, 5, 30 min treatment with LIF or 30 min treatment with serum. The phospho-proteins indicated were detected by immunoblotting of the same membrane, which had been cut referring to the size of the protein marker, then stripped and re-probed with the corresponding pan antibody. Results generated from the same blots are grouped, with each series terminating with the respective Gapdh as loading control.</p

iPSCs are less sensitive to LIF withdrawal than ESCs.

<p>ESCs or iPSCs were plated at a concentration of 8000 cells/cm², harvested after 4 days and analyzed for Nanog-GFP expression by flow cytometry. Cells were grown in KO SR (<b>A</b>), KO SR supplemented with 10% (v/v) Hyclone serum (HY) (<b>B</b>) or GMEM supplemented with HY (<b>C</b>) in presence or absence of LIF, as indicated. Numbers shown on the histograms are the mean of FL1 fluorescence intensity (MFI). One representative experiment out of four is shown. <b>D</b> ESCs or iPSCs were seeded at 8500 cells/cm² in KO SR in the presence or absence of LIF. Protein extracts were prepared after 4 days incubation and immunoblotting performed with the antibodies indicated. Following detection of pY705 Stat-3, blots were stripped and reprobed to detect total levels of Stat-3. <b>E</b> and <b>F</b> Cells were seeded at 8500 cells/cm² in KO SR plus 10% (v/v) HY (<b>E</b>) or GMEM supplemented with 10% (v/v) HY (<b>F</b>), in the presence or absence of LIF. After 48 h protein extracts were prepared and immunoblotting performed as indicated.</p