bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2019‒09‒29
twenty-one papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute


  1. J Biol Chem. 2019 Sep 22. pii: jbc.RA119.010036. [Epub ahead of print]
    Kearney AL, Cooke KC, Norris DM, Zadoorian A, Krycer JR, Fazakerley DJ, Burchfield JG, James DE.
      The Ser/Thr protein kinase Akt regulates essential biological processes such as cell survival, growth, and metabolism. Upon growth factor stimulation Akt is phosphorylated at Ser474, however how this phosphorylation contributes to Akt activation remains controversial. Previous studies, which induced loss of Ser474 phosphorylation by ablating its upstream kinase mTORC2, have implicated Ser474 phosphorylation as a driver of Akt substrate specificity. Here, we directly studied the role of Akt2 Ser474 phosphorylation in 3T3-L1 adipocytes by preventing Ser474 phosphorylation, without perturbing mTORC2 activity. This was achieved by utilising a chemical genetics approach, whereby ectopically expressed Ser474Ala Akt2 was engineered with a Trp80Ala mutation to confer resistance to the Akt inhibitor MK2206, and thus allow for its activation independently of endogenous Akt. We found that insulin-stimulated phosphorylation of four bona fide Akt substrates (TSC2, PRAS40, FOXO1/3a and AS160) were reduced by approximately 50% in the absence of Ser474 phosphorylation. Accordingly, insulin-stimulated mTORC1 activation, protein synthesis, FOXO nuclear exclusion, GLUT4 translocation and glucose uptake were attenuated upon loss of Ser474 phosphorylation. We propose a model whereby Ser474 phosphorylation is required for maximal Akt2 kinase activity in adipocytes.
    Keywords:  Akt PKB; Akt S474 phosphorylation; Akt W80A; GLUT4; MK2206; adipocyte; cell signaling; chemical genetics; glucose transport; insulin; mTOR complex (mTORC); phosphorylation; protein synthesis; serine/threonine protein kinase; substrate specificity
    DOI:  https://doi.org/10.1074/jbc.RA119.010036
  2. NPJ Breast Cancer. 2019 ;5 31
    Nixon MJ, Formisano L, Mayer IA, Estrada MV, González-Ericsson PI, Isakoff SJ, Forero-Torres A, Won H, Sanders ME, Solit DB, Berger MF, Cantley LC, Winer EP, Arteaga CL, Balko JM.
      Clinical trials have demonstrated the efficacy of combining phosphoinositide 3-kinase (PI3K) inhibitors with endocrine therapies in hormone therapy-refractory breast cancer. However, biomarkers of PI3K pathway dependence in ER+ breast cancer have not been fully established. Hotspot mutations in the alpha isoform of PI3K (PIK3CA) are frequent in ER+ disease and may identify tumors that respond to PI3K inhibitors. It is unclear whether PIK3CA mutations are the only biomarker to suggest pathway dependence and response to therapy. We performed correlative molecular characterization of primary and metastatic tissue from patients enrolled in a phase Ib study combining buparlisib (NVP-BKM-120), a pan-PI3K inhibitor, with letrozole in ER+, human epidermal growth factor-2 (HER2)-negative, metastatic breast cancer. Activating mutations in PIK3CA and inactivating MAP3K1 mutations marked tumors from patients with clinical benefit (≥6 months of stable disease). Patients harboring mutations in both genes exhibited the greatest likelihood of clinical benefit. In ER+ breast cancer cell lines, siRNA-mediated knockdown of MAP3K1 did not affect the response to buparlisib. In a subset of patients treated with buparlisib or the PI3Kα inhibitor alpelisib each with letrozole where PAM50 analysis was performed, nearly all tumors from patients with clinical benefit had a luminal A subtype. Mutations in MAP3K1 in ER+ breast cancer may be associated with clinical benefit from combined inhibition of PI3K and ER, but we could not ascribe direct biological function therein, suggesting they may be a surrogate for luminal A status. We posit that luminal A tumors may be a target population for this therapeutic combination.
    Keywords:  Cancer genomics; Tumour biomarkers
    DOI:  https://doi.org/10.1038/s41523-019-0126-6
  3. Nat Commun. 2019 Sep 24. 10(1): 4332
    Wu RC, Young IC, Chen YF, Chuang ST, Toubaji A, Wu MY.
      PTEN is frequently mutated in prostate cancer. The tumor suppressor function of PTEN is attributed to its lipid phosphatase activity that counters PI3K action. Here, we report a PTEN-ARID4B-PI3K axis in which PTEN inhibits expression of ARID4B, while ARID4B is a transcriptional activator of the PI3K subunit genes PIK3CA and PIK3R2 that are crucial for activation of the PI3K/AKT pathway. Reciprocal binding of ARID4B and histone H1 to the PIK3CA and PIK3R2 promoters modulates chromatin condensation, suggesting a mechanism by which ARID4B activates these promoters. Functional analyses reveals that ARID4B is required for prostate tumorigenesis when PTEN is deficient. The biological significance is further substantiated by the existence of a PTEN/ARID4B/PIK3CA three-gene signature that improves the predictive power for prostate cancer recurrence in patients. In summary, we identify ARID4B as a master regulator in the PTEN-PI3K pathway, thus providing a potential therapeutic target for prostate cancer carrying PTEN mutations.
    DOI:  https://doi.org/10.1038/s41467-019-12184-8
  4. Nat Cell Biol. 2019 Sep 23.
    Lim CY, Davis OB, Shin HR, Zhang J, Berdan CA, Jiang X, Counihan JL, Ory DS, Nomura DK, Zoncu R.
      Cholesterol activates the master growth regulator, mTORC1 kinase, by promoting its recruitment to the surface of lysosomes by the Rag guanosine triphosphatases (GTPases). The mechanisms that regulate lysosomal cholesterol content to enable mTORC1 signalling are unknown. Here, we show that oxysterol binding protein (OSBP) and its anchors at the endoplasmic reticulum (ER), VAPA and VAPB, deliver cholesterol across ER-lysosome contacts to activate mTORC1. In cells lacking OSBP, but not other VAP-interacting cholesterol carriers, the recruitment of mTORC1 by the Rag GTPases is inhibited owing to impaired transport of cholesterol to lysosomes. By contrast, OSBP-mediated cholesterol trafficking drives constitutive mTORC1 activation in a disease model caused by the loss of the lysosomal cholesterol transporter, Niemann-Pick C1 (NPC1). Chemical and genetic inactivation of OSBP suppresses aberrant mTORC1 signalling and restores autophagic function in cellular models of Niemann-Pick type C (NPC). Thus, ER-lysosome contacts are signalling hubs that enable cholesterol sensing by mTORC1, and targeting the sterol-transfer activity of these signalling hubs could be beneficial in patients with NPC.
    DOI:  https://doi.org/10.1038/s41556-019-0391-5
  5. Cells. 2019 Sep 12. pii: E1075. [Epub ahead of print]8(9):
    Wei X, Luo L, Chen J.
      The mammalian target of rapamycin (mTOR), is a serine/threonine protein kinase and belongs to the phosphatidylinositol 3-kinase (PI3K)-related kinase (PIKK) family. mTOR interacts with other subunits to form two distinct complexes, mTORC1 and mTORC2. mTORC1 coordinates cell growth and metabolism in response to environmental input, including growth factors, amino acid, energy and stress. mTORC2 mainly controls cell survival and migration through phosphorylating glucocorticoid-regulated kinase (SGK), protein kinase B (Akt), and protein kinase C (PKC) kinase families. The dysregulation of mTOR is involved in human diseases including cancer, cardiovascular diseases, neurodegenerative diseases, and epilepsy. Tissue damage caused by trauma, diseases or aging disrupt the tissue functions. Tissue regeneration after injuries is of significance for recovering the tissue homeostasis and functions. Mammals have very limited regenerative capacity in multiple tissues and organs, such as the heart and central nervous system (CNS). Thereby, understanding the mechanisms underlying tissue regeneration is crucial for tissue repair and regenerative medicine. mTOR is activated in multiple tissue injuries. In this review, we summarize the roles of mTOR signaling in tissue regeneration such as neurons, muscles, the liver and the intestine.
    Keywords:  intestine; liver; mTOR signaling; metabolism; muscle; neuron; tissue regeneration
    DOI:  https://doi.org/10.3390/cells8091075
  6. Cancers (Basel). 2019 Sep 24. pii: E1423. [Epub ahead of print]11(10):
    Samocha A, Doh H, Kessenbrock K, Roose JP.
      Fluidity in cell fate or heterogeneity in cell identity is an interesting cell biological phenomenon, which at the same time poses a significant obstacle for cancer therapy. The mammary gland seems a relatively straightforward organ with stromal cells and basal- and luminal- epithelial cell types. In reality, the epithelial cell fates are much more complex and heterogeneous, which is the topic of this review. Part of the complexity comes from the dynamic nature of this organ: the primitive epithelial tree undergoes extensively remodeling and expansion during puberty, pregnancy, and lactation and, unlike most other organs, the bulk of mammary gland development occurs late, during puberty. An active cell biological debate has focused on lineage commitment to basal- and luminal- epithelial cell fates by epithelial progenitor and stem cells; processes that are also relevant to cancer biology. In this review, we discuss the current understanding of heterogeneity in mammary gland and recent insights obtained through lineage tracing, signaling assays, and organoid cultures. Lastly, we relate these insights to cancer and ongoing efforts to resolve heterogeneity in breast cancer with single-cell RNAseq approaches.
    Keywords:  3D cultures; breast cancer; cell fate; heterogeneity; mammary gland; organoids; signaling; single-cell RNAseq
    DOI:  https://doi.org/10.3390/cancers11101423
  7. Front Cell Dev Biol. 2019 ;7 180
    Mnatsakanyan H, Sabater I Serra R, Salmeron-Sanchez M, Rico P.
      Embryonic stem cells (ESCs) possess remarkable abilities, as they can differentiate into all cell types (pluripotency) and be self-renewing, giving rise to two identical cells. These characteristics make ESCs a powerful research tool in fundamental embryogenesis as well as candidates for use in regenerative medicine. Significant efforts have been devoted to developing protocols to control ESC fate, including soluble and complex cocktails of growth factors and small molecules seeking to activate/inhibit key signaling pathways for the maintenance of pluripotency states or activate differentiation. Here we describe a novel method for the effective maintenance of mouse ESCs, avoiding the supplementation of complex inhibitory cocktails or cytokines, e.g., LIF. We show that the addition of zinc to ESC cultures leads to a stable pluripotent state that shares biochemical, transcriptional and karyotypic features with the classical LIF treatment. We demonstrate for the first time that ESCs maintained in long-term cultures with added zinc, are capable of sustaining a stable ESCs pluripotent phenotype, as well as differentiating efficiently upon external stimulation. We show that zinc promotes long-term ESC self-renewal (>30 days) via activation of ZIP7 and AKT signaling pathways. Furthermore, the combination of zinc with LIF results in a synergistic effect that enhances LIF effects, increases AKT and STAT3 activity, promotes the expression of pluripotency regulators and avoids the expression of differentiation markers.
    Keywords:  AKT; ZIP7; embryonic stem cells (ESC); stemness maintenance; zinc
    DOI:  https://doi.org/10.3389/fcell.2019.00180
  8. Mol Cancer Res. 2019 Sep 23. pii: molcanres.0545.2019. [Epub ahead of print]
    Chandrasekaran S, Sasaki M, Scharer CD, Kissick HT, Patterson DG, Magliocca KR, Seykora JT, Sapkota B, Gutman DA, Cooper LA, Lesinski GB, Waller EK, Thomas SN, Kotenko SV, Boss JM, Moreno CS, Swerlick RA, Pollack BP.
      Molecular events activating the phosphoinositide-3 kinase (PI3K) pathway are frequently detected in human tumors and the activation of PI3K signaling alters numerous cellular processes including tumor cell proliferation, survival, and motility. More recent studies have highlighted the impact of PI3K signaling on the cellular response to interferons and other immunologic processes relevant to anti-tumor immunity. Given the ability of interferon (IFN)-γ to regulate antigen processing and presentation and the pivotal role of major histocompatibility complex (MHC) class I (MHCI) and II (MHCII) expression in T-cell mediated anti-tumor immunity, we sought to determine the impact of PI3K signaling on MHCI and MHCII induction by IFN-γ. We found that the induction of cell surface MHCI and MHCII molecules by IFN-γ is enhanced by the clinical grade PI3K inhibitors dactolisib and pictilisib. We also found that PI3K inhibition increases STAT1 protein levels following IFN-γ treatment and increases accessibility at genomic STAT1 binding motifs. Conversely, we found that pharmacologic activation of PI3K signaling can repress the induction of MHCI and MHCII molecules by IFN-γ and likewise the loss of PTEN attenuates the induction of MHCI, MHCII, and STAT1 by IFN-γ. Consistent with these in vitro studies, we found that within human head and neck squamous cell carcinomas, intra-tumoral regions with high phospho-AKT immunohistochemical (IHC) staining had reduced MHCI IHC staining. Implications: Collectively, these findings demonstrate that MHC expression can be modulated by PI3K signaling and suggest that activation of PI3K signaling may promote immune escape via effects on antigen presentation.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-19-0545
  9. FASEB J. 2019 Sep 27. fj201900722R
    Nascimento-Filho CHV, Webber LP, Borgato GB, Goloni-Bertollo EM, Squarize CH, Castilho RM.
      Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide and is characterized by a fast-paced growth. Like other solid tumors, the HNSCC growth rate results in the development of hypoxic regions identified by the expression of hypoxia-inducible factor 1α (HIF-1α). Interestingly, clinical data have shown that pharmacological induction of intratumoral hypoxia caused an unexpected rise in tumor metastasis and the accumulation of cancer stem cells (CSCs). However, little is known on the molecular circuitries involved in the presence of intratumoral hypoxia and the augmented population of CSCs. Here we explore the impact of hypoxia on the behavior of HNSCC and define that the controlling function of phosphatase and tensin homolog (PTEN) over HIF-1α expression and CSC accumulation are de-regulated during hypoxic events. Our findings indicate that hypoxic niches are poised to accumulate CSCs in a molecular process driven by the loss of PTEN activity. Furthermore, our data suggest that targeted therapies aiming at the PTEN/PI3K signaling may constitute an effective strategy to counteract the development of intratumoral hypoxia and the accumulation of CSCs.-Nascimento-Filho, C. H. V., Webber, L. P., Borgato, G. B., Goloni-Bertollo, E. M., Squarize, C. H., Castilho, R. M. Hypoxic niches are endowed with a protumorigenic mechanism that supersedes the protective function of PTEN.
    Keywords:  EMT; ROS; cancer stem cell; epithelial-mesenchymal transition; mTOR
    DOI:  https://doi.org/10.1096/fj.201900722R
  10. Cell Rep. 2019 Sep 24. pii: S2211-1247(19)31121-0. [Epub ahead of print]28(13): 3406-3422.e7
    Li Q, Zhao Q, Zhang J, Zhou L, Zhang W, Chua B, Chen Y, Xu L, Li P.
      Insulin-stimulated hepatic glycogen synthesis is central to glucose homeostasis. Here, we show that PPP1R3G, a regulatory subunit of protein phosphatase 1 (PP1), is directly phosphorylated by AKT. PPP1R3G phosphorylation fluctuates with fasting-refeeding cycle and is required for insulin-stimulated dephosphorylation, i.e., activation of glycogen synthase (GS) in hepatocytes. In this study, we demonstrate that knockdown of PPP1R3G significantly inhibits insulin response. The introduction of wild-type PPP1R3G, and not phosphorylation-defective mutants, increases hepatic glycogen deposition, blood glucose clearance, and insulin sensitivity in vivo. Mechanistically, phosphorylated PPP1R3G displays increased binding for, and promotes dephosphorylation of, phospho-GS. Furthermore, PPP1R3B, another regulatory subunit of PP1, binds to the dephosphorylated GS, thereby relaying insulin stimulation to hepatic glycogen deposition. Importantly, this PP1-mediated signaling cascade is independent of GSK3. Therefore, we reveal a regulatory axis consisting of insulin/AKT/PPP1R3G/PPP1R3B that operates in parallel to the GSK3-dependent pathway, controlling glycogen synthesis and glucose homeostasis in insulin signaling.
    DOI:  https://doi.org/10.1016/j.celrep.2019.08.066
  11. Nat Commun. 2019 Sep 27. 10(1): 4427
    Li Q, Fu J, Xia Y, Qi W, Ishikado A, Park K, Yokomizo H, Huang Q, Cai W, Rask-Madsen C, Kahn CR, King GL.
      Insulin and IGF-1 actions in vascular smooth muscle cells (VSMC) are associated with accelerated arterial intima hyperplasia and restenosis after angioplasty, especially in diabetes. To distinguish their relative roles, we delete insulin receptor (SMIRKO) or IGF-1 receptor (SMIGF1RKO) in VSMC and in mice. Here we report that intima hyperplasia is attenuated in SMIRKO mice, but not in SMIGF1RKO mice. In VSMC, deleting IGF1R increases homodimers of IR, enhances insulin binding, stimulates p-Akt and proliferation, but deleting IR decreases responses to insulin and IGF-1. Studies using chimeras of IR(extracellular domain)/IGF1R(intracellular-domain) or IGF1R(extracellular domain)/IR(intracellular-domain) demonstrate homodimer IRα enhances insulin binding and signaling which is inhibited by IGF1Rα. RNA-seq identifies hyaluronan synthase2 as a target of homo-IR, with its expression increases by IR activation in SMIGF1RKO mice and decreases in SMIRKO mice. Enhanced intima hyperplasia in diabetes is mainly due to insulin signaling via homo-IR, associated with increased Has2 expression.
    DOI:  https://doi.org/10.1038/s41467-019-12368-2
  12. Sci Signal. 2019 Sep 24. pii: eaaw8288. [Epub ahead of print]12(600):
    McFall T, Diedrich JK, Mengistu M, Littlechild SL, Paskvan KV, Sisk-Hackworth L, Moresco JJ, Shaw AS, Stites EC.
      Cancer treatment decisions are increasingly guided by which specific genes are mutated within each patient's tumor. For example, agents inhibiting the epidermal growth factor receptor (EGFR) benefit many colorectal cancer (CRC) patients, with the general exception of those whose tumor includes a KRAS mutation. However, among the various KRAS mutations, that which encodes the G13D mutant protein (KRASG13D) behaves differently; for unknown reasons, KRASG13D CRC patients benefit from the EGFR-blocking antibody cetuximab. Controversy surrounds this observation, because it contradicts the well-established mechanisms of EGFR signaling with regard to RAS mutations. Here, we identified a systems-level, mechanistic explanation for why KRASG13D cancers respond to EGFR inhibition. A computational model of RAS signaling revealed that the biophysical differences between the three most common KRAS mutants were sufficient to generate different sensitivities to EGFR inhibition. Integrated computation with experimentation then revealed a nonintuitive, mutant-specific dependency of wild-type RAS activation by EGFR that is determined by the interaction strength between KRAS and the tumor suppressor neurofibromin (NF1). KRAS mutants that strongly interacted with and competitively inhibited NF1 drove wild-type RAS activation in an EGFR-independent manner, whereas KRASG13D weakly interacted with and could not competitively inhibit NF1 and, thus, KRASG13D cells remained dependent on EGFR for wild-type RAS activity. Overall, our work demonstrates how systems approaches enable mechanism-based inference in genomic medicine and can help identify patients for selective therapeutic strategies.
    DOI:  https://doi.org/10.1126/scisignal.aaw8288
  13. Proc Natl Acad Sci U S A. 2019 Sep 23. pii: 201904774. [Epub ahead of print]
    Padi SKR, Singh N, Bearss JJ, Olive V, Song JH, Cardó-Vila M, Kraft AS, Okumura K.
      The Pim and AKT serine/threonine protein kinases are implicated as drivers of cancer. Their regulation of tumor growth is closely tied to the ability of these enzymes to mainly stimulate protein synthesis by activating mTORC1 (mammalian target of rapamycin complex 1) signaling, although the exact mechanism is not completely understood. mTORC1 activity is normally suppressed by amino acid starvation through a cascade of multiple regulatory protein complexes, e.g., GATOR1, GATOR2, and KICSTOR, that reduce the activity of Rag GTPases. Bioinformatic analysis revealed that DEPDC5 (DEP domain containing protein 5), a component of GATOR1 complex, contains Pim and AKT protein kinase phosphorylation consensus sequences. DEPDC5 phosphorylation by Pim and AKT kinases was confirmed in cancer cells through the use of phospho-specific antibodies and transfection of phospho-inactive DEPDC5 mutants. Consistent with these findings, during amino acid starvation the elevated expression of Pim1 overcame the amino acid inhibitory protein cascade and activated mTORC1. In contrast, the knockout of DEPDC5 partially blocked the ability of small molecule inhibitors against Pim and AKT kinases both singly and in combination to suppress tumor growth and mTORC1 activity in vitro and in vivo. In animal experiments knocking in a glutamic acid (S1530E) in DEPDC5, a phospho mimic, in tumor cells induced a significant level of resistance to Pim and the combination of Pim and AKT inhibitors. Our results indicate a phosphorylation-dependent regulatory mechanism targeting DEPDC5 through which Pim1 and AKT act as upstream effectors of mTORC1 to facilitate proliferation and survival of cancer cells.
    Keywords:  AKT kinase; DEPDC5; GATOR1; Pim kinase; mTORC1
    DOI:  https://doi.org/10.1073/pnas.1904774116
  14. Cancers (Basel). 2019 Sep 24. pii: E1422. [Epub ahead of print]11(10):
    Dossou AS, Basu A.
      Autophagy is a process of self-degradation that enables the cell to survive when faced with starvation or stressful conditions. The mechanistic target of rapamycin (mTOR), also known as the mammalian target of rapamycin, plays a critical role in maintaining a balance between cellular anabolism and catabolism. mTOR complex 1 (mTORC1) was unveiled as a master regulator of autophagy since inhibition of mTORC1 was required to initiate the autophagy process. Evidence has emerged in recent years to indicate that mTORC1 also directly regulates the subsequent steps of the autophagy process, including the nucleation, autophagosome elongation, autophagosome maturation and termination. By phosphorylating select protein targets of the autophagy core machinery and/or their regulators, mTORC1 can alter their functions, increase their proteasomal degradation or modulate their acetylation status, which is a key switch of the autophagy process. Moreover, it phosphorylates and alters the subcellular localization of transcription factors to suppress the expression of genes needed for autophagosome formation and lysosome biogenesis. The purpose of this review article is to critically analyze current literatures to provide an integrated view of how mTORC1 regulates various steps of the autophagy process.
    Keywords:  AMPK; ULK1; autophagosome maturation; autophagy initiation; autophagy regulation; elongation; mTORC1 substrates; macroautophagy; nucleation; transcriptional regulation
    DOI:  https://doi.org/10.3390/cancers11101422
  15. Nat Neurosci. 2019 Oct;22(10): 1624-1634
    Gazestani VH, Pramparo T, Nalabolu S, Kellman BP, Murray S, Lopez L, Pierce K, Courchesne E, Lewis NE.
      Hundreds of genes are implicated in autism spectrum disorder (ASD), but the mechanisms through which they contribute to ASD pathophysiology remain elusive. Here we analyzed leukocyte transcriptomics from 1- to 4-year-old male toddlers with ASD or typical development from the general population. We discovered a perturbed gene network that includes highly expressed genes during fetal brain development. This network is dysregulated in human induced pluripotent stem cell-derived neuron models of ASD. High-confidence ASD risk genes emerge as upstream regulators of the network, and many risk genes may impact the network by modulating RAS-ERK, PI3K-AKT and WNT-β-catenin signaling pathways. We found that the degree of dysregulation in this network correlated with the severity of ASD symptoms in the toddlers. These results demonstrate how the heterogeneous genetics of ASD may dysregulate a core network to influence brain development at prenatal and very early postnatal ages and, thereby, the severity of later ASD symptoms.
    DOI:  https://doi.org/10.1038/s41593-019-0489-x
  16. Mol Syst Biol. 2019 Sep;15(9): e8685
    Harton MD, Koh WS, Bunker AD, Singh A, Batchelor E.
      The p53 tumor suppressor regulates distinct responses to cellular stresses. Although different stresses generate different p53 dynamics, the mechanisms by which cells decode p53 dynamics to differentially regulate target genes are not well understood. Here, we determined in individual cells how canonical p53 target gene promoters vary in responsiveness to features of p53 dynamics. Employing a chemical perturbation approach, we independently modulated p53 pulse amplitude, duration, or frequency, and we then monitored p53 levels and target promoter activation in individual cells. We identified distinct signal processing features-thresholding in response to amplitude modulation, a refractory period in response to duration modulation, and dynamic filtering in response to frequency modulation. We then showed that the signal processing features not only affect p53 target promoter activation, they also affect p53 regulation and downstream cellular functions. Our study shows how different promoters can differentially decode features of p53 dynamics to generate distinct responses, providing insight into how perturbing p53 dynamics can be used to generate distinct cell fates.
    Keywords:  gene regulation; microfluidics; p53; single cell; transcription factor dynamics
    DOI:  https://doi.org/10.15252/msb.20188685
  17. Front Endocrinol (Lausanne). 2019 ;10 602
    Argente J, Tatton-Brown K, Lehwalder D, Pfäffle R.
      The second 360° European Meeting on Growth Hormone Disorders, held in Barcelona, Spain, in June 2017, included a session entitled Pragmatism vs. Curiosity in Genetic Diagnosis of Growth Disorders, which examined current concepts of genetics and growth in the clinical setting, in terms of both growth failure and overgrowth. For patients with short stature, multiple genes have been identified that result in GH deficiency, which may be isolated or associated with additional pituitary hormone deficiencies, or in growth hormone resistance, primary insulin-like growth factor (IGF) acid-labile subunit deficiency, IGF-I deficiency, IGF-II deficiency, IGF-I resistance, and primary PAPP-A2 deficiency. While genetic causes of short stature were previously thought to primarily be associated with the GH-IGF-I axis, it is now established that multiple genetic anomalies not associated with the GH-IGF-I axis can result in short stature. A number of genetic anomalies have also been shown to be associated with overgrowth, some of which involve the GH-IGF-I axis. In patients with overgrowth in combination with an intellectual disability, two predominant gene families, the epigenetic regulator genes, and PI3K/AKT pathway genes, have now been identified. Specific processes should be followed for decisions on which patients require genetic testing and which genes should be examined for anomalies. The decision to carry out genetic testing should be directed by the clinical process, not merely for research purposes. The intention of genetic testing should be to direct the clinical options for management of the growth disorder.
    Keywords:  diagnosis; genetics; growth hormone; overgrowth; short stature
    DOI:  https://doi.org/10.3389/fendo.2019.00602
  18. EMBO Rep. 2019 Sep 23. e47845
    Testini C, Smith RO, Jin Y, Martinsson P, Sun Y, Hedlund M, Sáinz-Jaspeado M, Shibuya M, Hellström M, Claesson-Welsh L.
      Exaggerated signaling by vascular endothelial growth factor (VEGF)-A and its receptor, VEGFR2, in pathologies results in poor vessel function. Still, pharmacological suppression of VEGFA/VEGFR2 may aggravate disease. Delineating VEGFR2 signaling in vivo provides strategies for suppression of specific VEGFR2-induced pathways. Three VEGFR2 tyrosine residues (Y949, Y1212, and Y1173) induce downstream signaling. Here, we show that knock-in of phenylalanine to create VEGFR2 Y1212F in C57Bl/6 and FVB mouse strains leads to loss of growth factor receptor-bound protein 2- and phosphoinositide 3'-kinase (PI3K)p85 signaling. C57Bl/6 Vegfr2Y1212F/Y1212F show reduced embryonic endothelial cell (EC) proliferation and partial lethality. FVB Vegfr2Y1212F/Y1212F show reduced postnatal EC proliferation. Reduced EC proliferation in Vegfr2Y1212F/Y1212F explants is rescued by c-Myc overexpression. We conclude that VEGFR2 Y1212 signaling induces activation of extracellular-signal-regulated kinase (ERK)1/2 and Akt pathways required for c-Myc-dependent gene regulation, endothelial proliferation, and vessel stability.
    Keywords:  GRB2; Nck; PI3Kp85; angiogenesis; proliferation
    DOI:  https://doi.org/10.15252/embr.201947845
  19. J Vis Exp. 2019 Sep 04.
    Chan K, Tong AHY, Brown KR, Mero P, Moffat J.
      Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.
    DOI:  https://doi.org/10.3791/59780
  20. Mol Cell. 2019 Sep 13. pii: S1097-2765(19)30659-8. [Epub ahead of print]
    Chung M, Liu C, Yang HW, Köberlin MS, Cappell SD, Meyer T.
      Cells escape the need for mitogens at a restriction point several hours before entering S phase. The restriction point has been proposed to result from CDK4/6 initiating partial Rb phosphorylation to trigger a bistable switch whereby cyclin E-CDK2 and Rb mutually reinforce each other to induce Rb hyperphosphorylation. Here, using single-cell analysis, we unexpectedly found that cyclin E/A-CDK activity can only maintain Rb hyperphosphorylation starting at the onset of S phase and that CDK4/6 activity, but not cyclin E/A-CDK activity, is required to hyperphosphorylate Rb throughout G1 phase. Mitogen removal in G1 results in a gradual loss of CDK4/6 activity with a high likelihood of cells sustaining Rb hyperphosphorylation until S phase, at which point cyclin E/A-CDK activity takes over. Thus, it is short-term memory, or transient hysteresis, in CDK4/6 activity following mitogen removal that sustains Rb hyperphosphorylation, demonstrating a probabilistic rather than an irreversible molecular mechanism underlying the restriction point.
    Keywords:  CDK; E2F; bistability; cell cycle; cyclin; hysteresis; restriction point; retinoblastoma
    DOI:  https://doi.org/10.1016/j.molcel.2019.08.020
  21. Proc Natl Acad Sci U S A. 2019 Sep 23. pii: 201905722. [Epub ahead of print]
    Tomasetti C, Poling J, Roberts NJ, London NR, Pittman ME, Haffner MC, Rizzo A, Baras A, Karim B, Kim A, Heaphy CM, Meeker AK, Hruban RH, Iacobuzio-Donahue CA, Vogelstein B.
      A new evaluation of previously published data suggested to us that the accumulation of mutations might slow, rather than increase, as individuals age. To explain this unexpected finding, we hypothesized that normal stem cell division rates might decrease as we age. To test this hypothesis, we evaluated cell division rates in the epithelium of human colonic, duodenal, esophageal, and posterior ethmoid sinonasal tissues. In all 4 tissues, there was a significant decrease in cell division rates with age. In contrast, cell division rates did not decrease in the colon of aged mice, and only small decreases were observed in their small intestine or esophagus. These results have important implications for understanding the relationship between normal stem cells, aging, and cancer. Moreover, they provide a plausible explanation for the enigmatic age-dependent deceleration in cancer incidence in very old humans but not in mice.
    Keywords:  aging; cancer; cell division; mutation rate
    DOI:  https://doi.org/10.1073/pnas.1905722116