Rb Family Research Articles

Distinct Cell-Cycle Control in Two Different States of Mouse Pluripotency

SummaryMouse embryonic stem cells (ESCs) cultured in serum are characterized by hyper-phosphorylated RB protein, lack of G1 control, and rapid progression through the cell cycle. Here, we show that ESCs grown in the presence of two small-molecule inhibitors (2i ESCs) have a longer G1-phase with hypo-phosphorylated RB, implying that they have a functional G1 checkpoint. Deletion of RB, P107, and P130 in 2i ESCs results in a G1-phase similar to that of serum ESCs. Inhibition of the ERK signaling pathway in serum ESCs results in the appearance of hypo-phosphorylated RB and the reinstatement of a G1 checkpoint. In addition, induction of a dormant state by the inhibition of MYC, resembling diapause, requires the presence of the RB family proteins. Collectively, our data show that RB-dependent G1 restriction point signaling is active in mouse ESCs grown in 2i but abrogated in serum by ERK-dependent phosphorylation.

Dnmt1-dependent Chk1 pathway suppression is protective against neuron division.

Neuronal differentiation and cell-cycle exit are tightly coordinated, even in pathological situations. When pathological neurons re-enter the cell cycle and progress through the S phase, they undergo cell death instead of division. However, the mechanisms underlying mitotic resistance are mostly unknown. Here, we have found that acute inactivation of retinoblastoma (Rb) family proteins (Rb, p107 and p130) in mouse postmitotic neurons leads to cell death after S-phase progression. Checkpoint kinase 1 (Chk1) pathway activation during the S phase prevented the cell death, and allowed the division of cortical neurons that had undergone acute Rb family inactivation, oxygen-glucose deprivation (OGD) or in vivo hypoxia-ischemia. During neurogenesis, cortical neurons became protected from S-phase Chk1 pathway activation by the DNA methyltransferase Dnmt1, and underwent cell death after S-phase progression. Our results indicate that Chk1 pathway activation overrides mitotic safeguards and uncouples neuronal differentiation from mitotic resistance.

Rb-independent E2F3 promotes cell proliferation and alters expression of genes involved in metabolism and inflammation.

E2F transcription factors are key targets of the retinoblastoma (Rb) tumor suppressor. Despite extensive studies, the in vivo consequences of disrupting the interaction between Rb and an individual E2F are not clear. Here, we report an E2F mutation that interfered with binding to Rb family proteins without significantly affecting protein level or transactivation function. Characterization of mouse embryonic fibroblasts with this Rb‐independent E2F3LQ mutation revealed that disrupting the Rb and E2F3 interaction increased cell proliferation, allowed cells to accumulate to higher density, and significantly altered expression of genes involved in metabolism, inflammation, immunity, and response to stress. These results suggest that the Rb‐independent E2FLQ mutations might provide useful tools to investigate the in vivo consequences of disrupting the interactions between Rb and E2F.

Correction: Ablating all three retinoblastoma family members in mouse lung leads to neuroendocrine tumor formation.

// Sara Lazaro 1 , Miriam Perez-Crespo 1 , Ana Belen Enguita 2 , Pilar Hernandez 1 , Jesus Martinez-Palacio 1 , Marta Oteo 3 , Julien Sage 4, 5 , Jesus M. Paramio 1, 6, 7 , Mirentxu Santos 1, 6, 7 1 Molecular Oncology Unit Institute of Biomedical Investigation University Hospital “12 de Octubre”, Madrid, Spain 2 Pathology Department Institute of Biomedical Investigation University Hospital “12 de Octubre”, Madrid, Spain 3 Biomedical Applications and Pharmacokinetics CIEMAT (ed 70A), Madrid, Spain 4 Department of Pediatrics, Stanford University, Stanford, USA 5 Department of Genetics. Stanford University, CCSR Rm. 1215a. Stanford, California, USA 6 Molecular Oncology, Institute of Biomedical Investigation University Hospital “12 de Octubre”, Madrid, Spain 7 Centro de Investigaciones Biomedicas en Red de Cancer (CIBERONC), Madrid, Spain Correspondence to: Jesus M. Paramio, email: jesusm.paramio@ciemat.es Mirentxu Santos, email: mirentxu.santos@ciemat.es Keywords: retinoblastoma family, lung cancer, urethane, DHPN Received: January 13, 2016 Accepted: December 05, 2016 Published: December 10, 2016 ABSTRACT Lung cancer is a deadly disease with increasing cases diagnosed worldwide and still a very poor prognosis. While mutations in the retinoblastoma ( RB1 ) tumor suppressor have been reported in lung cancer, mainly in small cell lung carcinoma, the tumor suppressive role of its relatives p107 and p130 is still a matter of debate. To begin to investigate the role of these two Rb family proteins in lung tumorigenesis, we have generated a conditional triple knockout mouse model (TKO) in which the three Rb family members can be inactivated in adult mice. We found that ablation of all three family members in the lung of mice induces tumorlets, benign neuroendocrine tumors that are remarkably similar to their human counterparts. Upon chemical carcinogenesis, DHPN and urethane accelerate tumor development; the TKO model displays increased sensitivity to DHPN, and urethane increases malignancy of tumors. All the tumors developing in TKO mice (spontaneous and chemically induced) have neuroendocrine features but do not progress to fully malignant tumors. Thus, loss of Rb and its family members confers partial tumor susceptibility in neuroendocrine lineages in the lungs of mice. Our data also imply the requirement of other oncogenic signaling pathways to achieve full transformation in neuroendocrine lung lesions mutant for the Rb family.

Rb family proteins enforce the homeostasis of quiescent hematopoietic stem cells by repressing Socs3 expression.

Prolonged exit from quiescence by hematopoietic stem cells (HSCs) progressively impairs their homeostasis in the bone marrow through an unidentified mechanism. We show that Rb proteins, which are major enforcers of quiescence, maintain HSC homeostasis by positively regulating thrombopoietin (Tpo)-mediated Jak2 signaling. Rb family protein inactivation triggers the progressive E2f-mediated transactivation of Socs3, a potent inhibitor of Jak2 signaling, in cycling HSCs. Aberrant activation of Socs3 impairs Tpo signaling and leads to impaired HSC homeostasis. Therefore, Rb proteins act as a central hub of quiescence and homeostasis by coordinating the regulation of both cell cycle and Jak2 signaling in HSCs.

Genomic profiles of low-grade murine gliomas evolve during progression to glioblastoma.

Gliomas are diverse neoplasms with multiple molecular subtypes. How tumor-initiating mutations relate to molecular subtypes as these tumors evolve during malignant progression remains unclear. We used genetically engineered mouse models, histopathology, genetic lineage tracing, expression profiling, and copy number analyses to examine how genomic tumor diversity evolves during the course of malignant progression from low- to high-grade disease. Knockout of all 3 retinoblastoma (Rb) family proteins was required to initiate low-grade tumors in adult mouse astrocytes. Mutations activating mitogen-activated protein kinase signaling, specifically KrasG12D, potentiated Rb-mediated tumorigenesis. Low-grade tumors showed mutant Kras-specific transcriptome profiles but lacked copy number mutations. These tumors stochastically progressed to high-grade, in part through acquisition of copy number mutations. High-grade tumor transcriptomes were heterogeneous and consisted of 3 subtypes that mimicked human mesenchymal, proneural, and neural glioblastomas. Subtypes were confirmed in validation sets of high-grade mouse tumors initiated by different driver mutations as well as human patient-derived xenograft models and glioblastoma tumors. These results suggest that oncogenic driver mutations influence the genomic profiles of low-grade tumors and that these, as well as progression-acquired mutations, contribute strongly to the genomic heterogeneity across high-grade tumors.

Human cytomegalovirus-encoded viral cyclin-dependent kinase (v-CDK) UL97 phosphorylates and inactivates the retinoblastoma protein-related p107 and p130 proteins

The human cytomegalovirus (HCMV)-encoded viral cyclin-dependent kinase (v-CDK) UL97 phosphorylates the retinoblastoma (Rb) tumor suppressor. Here, we identify the other Rb family members p107 and p130 as novel targets of UL97. UL97 phosphorylates p107 and p130 thereby inhibiting their ability to repress the E2F-responsive E2F1 promoter. As with Rb, this phosphorylation, and the rescue of E2F-responsive transcription, is dependent on the L1 LXCXE motif in UL97 and its interacting clefts on p107 and p130. Interestingly, UL97 does not induce the disruption of all p107-E2F or p130-E2F complexes, as it does to Rb-E2F complexes. UL97 strongly interacts with p107 but not Rb or p130. Thus the inhibitory mechanisms of UL97 for Rb family protein-mediated repression of E2F-responsive transcription appear to differ for each of the Rb family proteins. The immediate early 1 (IE1) protein of HCMV also rescues p107- and p130-mediated repression of E2F-responsive gene expression, but it does not induce their phosphorylation and does not disrupt p107-E2F or p130-E2F complexes. The unique regulation of Rb family proteins by HCMV UL97 and IE1 attests to the importance of modulating Rb family protein function in HCMV-infected cells.

Highlights of This Issue

Metabolomics captures the interactions between genetic alterations, enzymatic activity, and metabolic reactions. Here, comparative mass spectrometry-based metabolomics was performed using matched frozen and fixed isogenic prostate cancer cells and clinical specimens. A sufficient number of metabolites were retained in formalin-fixed, paraffin-embedded (FFPE) tissue to allow unsupervised hierarchical clustering of cell populations. This metabolomic profiling approach creates the opportunity, for research and diagnostic purposes, to interrogate annotated tumor banks retrospectively and link metabolomics with clinicopathologic endpoints. Combined, metabolic profiling to discover and validate metabolism-based diagnostic, prognostic or predictive biomarkers in archival diagnostic tissue adds a new, powerful technology for precision medicine.A critical aspect of metastatic epithelial ovarian cancer (EOC) is tumor cell dormancy, rendering these cells resistant to chemotherapeutics. Employing a systematic approach to analyze the role of negative growth factors, MacDonald and colleagues demonstrate that disruption of the DP, RB family, E2F, and MuvB complex (DREAM) results in death of EOC cells. Specifically, loss of the DREAM assembly factor Dyrk1A, or the DREAM component p130, impairs repression of DREAM target genes, leads to continued DNA synthesis, and is coincident with increased cell death. Importantly, chemical inhibition of Dyrk1A synergizes with conventional chemotherapies to kill EOC cells under dormant conditions.The basis of resistance or sensitivity to irinotecan in colorectal cancer has still not been clearly established. Strategies to identify susceptible patients and targetable pathways to reverse resistance are urgently needed. Cabal-Hierro and O’Dwyer highlight a role for RIP1 kinase in SN38 sensitivity by mediating cell death/DNA damage signaling in colon adenocarcinoma model systems through the TNF/TNFR pathway. Downregulation of RIP1 protects from SN-38 in vitro and in vivo, suggesting that RIP1 has potential as a biomarker. Activation of TNF/TNFR signaling potentiates SN38 activity in a RIP1-dependent manner; thus, suggesting a re-evaluation of TNF-based interventions to enhance treatment efficacy.Early metastasis and therapeutic resistance are huge setbacks to patients diagnosed with pancreatic ductal adenocarcinoma (PDAC). Here, Smigiel and colleagues report a unique function of the tumor microenvironment (TME) cytokine oncostatin M (OSM) in inducing epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties in pancreatic cancer cells. Elevated levels of OSM in PDAC induce EMT transcription factors ZEB1 and Snail, and induce CD44+ CSC that are more migratory, tumorigenic, highly metastatic and resistant to gemcitabine. As such, targeting OSM or OSM-signaling in PDAC is a potential therapeutic strategy to prevent de novo acquisition of CSC properties such as drug resistance and metastasis.

Decreased transcriptional corepressor p107 is associated with exercise-induced mitochondrial biogenesis in human skeletal muscle.

Increased mitochondrial content is a hallmark of exercise‐induced skeletal muscle remodeling. For this process, considerable evidence underscores the involvement of transcriptional coactivators in mediating mitochondrial biogenesis. However, our knowledge regarding the role of transcriptional corepressors is lacking. In this study, we assessed the association of the transcriptional corepressor Rb family proteins, Rb and p107, with endurance exercise‐induced mitochondrial adaptation in human skeletal muscle. We showed that p107, but not Rb, protein levels decrease by 3 weeks of high‐intensity interval training. This is associated with significant inverse association between p107 and exercise‐induced improved mitochondrial oxidative phosphorylation. Indeed, p107 showed significant reciprocal correlations with the protein contents of representative markers of mitochondrial electron transport chain complexes. These findings in human skeletal muscle suggest that attenuated transcriptional repression through p107 may be a novel mechanism by which exercise stimulates mitochondrial biogenesis following exercise.

RB inactivation in keratin 18 positive thymic epithelial cells promotes non-cell autonomous T cell hyperproliferation in genetically engineered mice

Thymic epithelial cells (TEC), as part of thymic stroma, provide essential growth factors/cytokines and self-antigens to support T cell development and selection. Deletion of Rb family proteins in adult thymic stroma leads to T cell hyperplasia in vivo. To determine whether deletion of Rb specifically in keratin (K) 18 positive TEC was sufficient for thymocyte hyperplasia, we conditionally inactivated Rb and its family members p107 and p130 in K18+ TEC in genetically engineered mice (TgK18GT121; K18 mice). We found that thymocyte hyperproliferation was induced in mice with Rb inactivation in K18+ TEC, while normal T cell development was maintained; suggesting that inactivation of Rb specifically in K18+ TEC was sufficient and responsible for the phenotype. Transplantation of wild type bone marrow cells into mice with Rb inactivation in K18+ TEC resulted in donor T lymphocyte hyperplasia confirming the non-cell autonomous requirement for Rb proteins in K18+ TEC in regulating T cell proliferation. Our data suggests that thymic epithelial cells play an important role in regulating lymphoid proliferation and thymus size.

RB: An essential player in adult neurogenesis.

The fundamental mechanisms underlying adult neurogenesis remain to be fully clarified. Members of the cell cycle machinery have demonstrated key roles in regulating adult neural stem cell (NSC) quiescence and the size of the adult-born neuronal population. The retinoblastoma protein, Rb, is known to possess CNS-specific requirements that are independent from its classical role as a tumor suppressor. The recent study by Vandenbosch etal. has clarified distinct requirements for Rb during adult neurogenesis, in the restriction of proliferation, as well as long-term adult-born neuronal survival. However, Rb is no longer believed to be the main cell cycle regulator maintaining the quiescence of adult NSCs. Future studies must consider Rb as part of a larger network of regulatory effectors, including the other members of the Rb family, p107 and p130. This will help elucidate the contribution of Rb and other pocket proteins in the context of adult neurogenesis, and define its crucial role in regulating the size and fate of the neurogenic niche.

Ablating all three retinoblastoma family members in mouse lung leads to neuroendocrine tumor formation.

Lung cancer is a deadly disease with increasing cases diagnosed worldwide and still a very poor prognosis. While mutations in the retinoblastoma (RB1) tumor suppressor have been reported in lung cancer, mainly in small cell lung carcinoma, the tumor suppressive role of its relatives p107 and p130 is still a matter of debate. To begin to investigate the role of these two Rb family proteins in lung tumorigenesis, we have generated a conditional triple knockout mouse model (TKO) in which the three Rb family members can be inactivated in adult mice. We found that ablation of all three family members in the lung of mice induces tumorlets, benign neuroendocrine tumors that are remarkably similar to their human counterparts. Upon chemical carcinogenesis, DHPN and urethane accelerate tumor development; the TKO model displays increased sensitivity to DHPN, and urethane increases malignancy of tumors. All the tumors developing in TKO mice (spontaneous and chemically induced) have neuroendocrine features but do not progress to fully malignant tumors. Thus, loss of Rb and its family members confers partial tumor susceptibility in neuroendocrine lineages in the lungs of mice. Our data also imply the requirement of other oncogenic signaling pathways to achieve full transformation in neuroendocrine lung lesions mutant for the Rb family.

Expression of the lncRNA Maternally Expressed Gene 3 (MEG3) Contributes to the Control of Lung Cancer Cell Proliferation by the Rb Pathway

Maternally expressed gene 3 (MEG3, mouse homolog Gtl2) encodes a long noncoding RNA (lncRNA) that is expressed in many normal tissues, but is suppressed in various cancer cell lines and tumors, suggesting it plays a functional role as a tumor suppressor. Hypermethylation has been shown to contribute to this loss of expression. We now demonstrate that MEG3 expression is regulated by the retinoblastoma protein (Rb) pathway and correlates with a change in cell proliferation. Microarray analysis of mouse embryonic fibroblasts (MEFs) isolated from mice with genetic deletion of all three Rb family members (TKO) revealed a significant silencing of Gtl2/MEG3 expression compared to WT MEFs, and re-expression of Gtl2/MEG3 caused decrease in cell proliferation and increased apoptosis. MEG3 levels also were suppressed in A549 lung cancer cells compared with normal human bronchial epithelial (NHBE) cells, and, similar to the TKO cells, re-constitution of MEG3 led to a decrease in cell proliferation and elevated apoptosis. Activation of pRb by treatment of A549 and SK-MES-1 cells with palbociclib, a CDK4/6 inhibitor, increased the expression of MEG3 in a dose-dependent manner, while knockdown of pRb/p107 attenuated this effect. In addition, expression of phosphorylation-deficient mutant of pRb increased MEG3 levels in both lung cancer cell types. Treatment of these cells with palbociclib also decreased the expression of pRb-regulated DNA methyltransferase 1 (DNMT1), while conversely, knockdown of DNMT1 resulted in increased expression of MEG3. As gene methylation has been suggested for MEG3 regulation, we found that palbociclib resulted in decreased methylation of the MEG3 locus similar to that observed with 5-aza-deoxycytidine. Anti-sense oligonucleotide silencing of drug-induced MEG3 expression in A549 and SK-MES-1 cells partially rescued the palbociclib-mediated decrease in cell proliferation, while analysis of the TCGA database revealed decreased MEG3 expression in human lung tumors harboring a disrupted RB pathway. Together, these data suggest that disruption of the pRb-DNMT1 pathway leads to a decrease in MEG3 expression, thereby contributing to the pro-proliferative state of certain cancer cells.

RB family proteins ensure the homeostasis of quiescent hematopoietic stem cells by preserving intracellular cytokine signaling

RB family proteins ensure the homeostasis of quiescent hematopoietic stem cells by preserving intracellular cytokine signaling

Abstract 3029: Dual targeting of ARK5 and CDK4 pathways with ON 123300 as a therapeutic strategy for colorectal carcinoma

Abstract Introduction: This study describes the development of a novel dual specificity kinase inhibitor, ON 123300, which exhibits potent activity against colorectal cancers both in vitro and in vivo. While overexpression of Cyclin D1 is closely correlated with the proliferative rate of these tumor cells, metastatic colorectal cancers over-express ARK5, a member of the AMPK family and mediator of AKT activation. In this study, we show that ON 123300, which inhibits both CDK4/6 and ARK5, is a potent inducer of apoptosis of colorectal cancer cells when compared to palbociclib, a highly selective inhibitor of CDK4/6 kinases that does not target ARK5. Results & Conclusions: We examined the effects of palbociclib and ON 123300 on cell cycle progression, modulation of Rb and PI3K/AKT pathways, and induction of apoptosis in multiple colorectal cancer cell lines. Comparative kinase inhibition assays showed that while palbociclib and ON 123300 exhibited equivalent inhibition against CDK4/CDK6, ARK5 activity was inhibited only by ON 123300. When DLD1 and SW480 cells were incubated with increasing concentrations of palbociclib or ON 123300, both compounds were equally efficient in their ability to inhibit phosphorylation of all three members of the Rb family of proteins. However, when the phosphorylation status of proteins associated with the PI3K/AKT pathway was measured by western blot, ON 123300 showed concentration-dependent inhibition of mTOR, AKT, 4EBP1 and S6RB phosphorylation while palbociclib had little or no effect on the phosphorylation of these proteins. Cells treated with palbociclib rapidly accumulated in the G0/G1 stage of the cell cycle with increasing drug concentrations. Although cells treated with ON 123300 also arrested in the G0/G1 phase at lower concentrations (01-0.5 uM), with increasing concentrations of drug there was an accumulation of cells with sub-G1 DNA content, suggesting induction of apoptosis. ON 123300-treated cells showed cleavage of PARP and Caspases (3, 7 and 9) as well as inhibition of FOXO1 phosphorylation, which was not observed in cells treated with palbociclib. Since ARK5 belongs to the AMPK family of kinases, we next examined the effects of ON 123300-mediated ARK5 inhibition on metabolic changes of tumor cells that over-express this gene. Treatment of SW-480 colorectal cancer cells with ON 123300 resulted in an increase in glucose uptake, profound inhibition of glutamine uptake and reduced ATP production. A detailed metabolomic study revealed significant alterations in the levels of metabolites associated with glutamine metabolism. Nude mouse xenograft assays using Colo-205 cells revealed strong inhibition of tumor growth following 100 mg/kg of ON 123300 given QD or QOD, with little evidence of toxicity as measured by change in body weight. Thus, dual inhibition of ARK5 and CDK4 pathways could be an effective therapeutic strategy for the treatment of colorectal cancers. Citation Format: Saikrishna A. Divakar, M.V. Ramana Reddy, Stephen C. Cosenza, Stacey J. Baker, Vinee Purohit, Venugopal Gunda, Pankaj K. Singh, E. Premkumar Reddy. Dual targeting of ARK5 and CDK4 pathways with ON 123300 as a therapeutic strategy for colorectal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3029.

Regulation of cell polarity determinants by the Retinoblastoma tumor suppressor protein.

In addition to their canonical roles in the cell cycle, RB family proteins regulate numerous developmental pathways, although the mechanisms remain obscure. We found that Drosophila Rbf1 associates with genes encoding components of the highly conserved apical–basal and planar cell polarity pathways, suggesting a possible regulatory role. Here, we show that depletion of Rbf1 in Drosophila tissues is indeed associated with polarity defects in the wing and eye. Key polarity genes aPKC, par6, vang, pk, and fmi are upregulated, and an aPKC mutation suppresses the Rbf1-induced phenotypes. RB control of cell polarity may be an evolutionarily conserved function, with important implications in cancer metastasis.

Cell division of neurons in an oxygen-glucose deprivation model

Event Abstract Back to Event Cell division of neurons in an oxygen-glucose deprivation model Itsuki Ajioka1, 2* and Mio Oshikawa1 1 Tokyo Medical and Dental University (TMDU), Center for Brain Integration Research (CBIR), Japan 2 JST, PRESTO, Japan Although neurons are able to re-enter the cell cycle in pathological situations such as neurodegeneration and stroke, they are tightly protected from cell division and undergo cell death after S phase progression. Neurons become permanently post-mitotic immediately after cell cycle exit during development, however, the mechanism underlying non-dividing feature is mostly unknown. The Retinoblastoma protein family (Rb, p107, p130) plays a central role in preventing cells from entering the S phase. When Rb family expression is lost in neuronal progenitor cells, the subsequent coordination of cell-cycle exit and neuronal differentiation is lost, and neurons can divide in some cases such as retinoblastoma (Ajioka et al., 2007). We recently developed a technique for conditionally inactivating all of the Rb family members in mouse cortical progenitors, either before or immediately after cell-cycle exit, by electroporation with Cre-expressing plasmids containing a ubiquitous pCAG promoter or a neuron-specific pMAP2 promoter (Oshikawa et al., 2013). When the Rb family is inactivated using pCAG-Cre, immature neurons generated from the pCAG-induced Rb-TKO (Rb-/-; p107-/-; p130-/-) progenitors divide. In contrast, the pMAP2-induced Rb-TKO immature neurons enter the S phase, but undergo cell death. Thus, once progenitor daughter cells exit the cell-cycle and initiate neuronal differentiation, they are prevented from undergoing cell division, and maintain mitotic resistance even after acute Rb family inactivation. These findings led us to hypothesize that pathological neurons in the S phase undergo cell death by activating mitotic safeguards. To investigate whether such mitotic safeguard protects pathological neurons in the S phase from undergoing cell division, we used oxygen-glucose deprivation (OGD) model in which post-mitotic cortical neurons undergo cell death after S phase progression and Rb phosphorylation. We demonstrate the cell division of dying OGD neurons in the S phase by inactivating mitotic safeguards. Keywords: Cell Cycle, Cell Division, Phosphorylation, Neuron, retinoblastoma protein family Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Poster Presentation Session Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Ajioka I and Oshikawa M (2016). Cell division of neurons in an oxygen-glucose deprivation model. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00182 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Prof. Itsuki Ajioka, Tokyo Medical and Dental University (TMDU), Center for Brain Integration Research (CBIR), Tokyo, Tokyo, Japan, iajioka.cbir@tmd.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Itsuki Ajioka Mio Oshikawa Google Itsuki Ajioka Mio Oshikawa Google Scholar Itsuki Ajioka Mio Oshikawa PubMed Itsuki Ajioka Mio Oshikawa Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Role of the RB-Interacting Proteins in Stem Cell Biology.

Human retinoblastoma gene RB1 is the first tumor suppressor gene (TSG) isolated by positional cloning in 1986. RB is extensively studied for its ability to regulate cell cycle by binding to E2F1 and inhibiting the transcriptional activity of the latter. In human embryonic stem cells (ESCs), only a minute trace of RB is found in complex with E2F1. Increased activity of RB triggers differentiation, cell cycle arrest, and cell death. On the other hand, inactivation of the entire RB family (RB1, RBL1, and RBL2) in human ESC induces G2/M arrest and cell death. These observations indicate that both loss and overactivity of RB could be lethal for the stemness of cells. A question arises why inactive RB is required for the survival and stemness of cells? To shed some light on this question, we analyzed the RB-binding proteins. In this review we have focused on 27 RB-binding partners that may have potential roles in different aspects of stem cell biology.

Differential Roles Between ERK1 and ERK2 on Sensitivity of Radiation Resistance Prostate Cancer Cells in Response to Ionizing Radiation

ERKs are critical regulators for cell proliferation and play an important role in prostate cancer (PCa). Previously, we selected from the DU145 cell line (DU) a subset of cells (DUIR) that survive fractionated high-dose ionizing radiation (IR) in vitro and have identified ERK1/2 signaling as the important regulator that determines IR resistance and IR-induced aggressiveness in PrCa cells. In this study we further study the different roles between ERK1 and ERK2 involving in the regulation of cell survival in response to IR. Cell growth and survival were assessed using cell counting, MTT assay, and clonogenic assay. Caspase activity and expression of cleaved PARP were determined for apoptosis. Activation of ERK and expression of biomarkers was assessed using immunoblots. Disruption of ERK1/2 signaling pathway was fulfilled by either using inhibitor U0126 or down-regulation of ERK1 and/or ERK2 with shRNA. Cell cycle was analyzed by using flow cytometry. DUIR has undergone epithelial-mesenchymal transition (EMT) as defined by reduced E-cadherin and enhanced vimentin expression. DUIR cells display higher levels of neurotensin receptor 1 (NTR1), neuron-specific enolase (NSE) and chromogranin B, resembling a neuroendocrine (NE) phenotype. DUIR is more resistant to IR (6-10 Gy) or to zinc-directed toxins than DU. ERK1/2 remain constitutively active in DUIR cells. Inhibition of ERK1/2 signaling by U0126 sensitizes DUIR cells to IR. To further assess the role of ERK1/2 in cell survivability to IR, we generated stable cell lines using lentivirus carrying ERK1- or ERK2-shRNA. Down-regulation of ERK1 and/or ERK2 sensitized the cells to IR, as shown by decreased proliferation, decreased clonogenicity, and increased apoptosis. Reduction of Bcl2 and bcl-x in DUIR cell upon IR is shown in ERK1 shRNA cells but not in ERK2-shRNA or control cells. Meanwhile, XIAP level in ERK2-shRNA cells upon IR is significantly reduced. p130, an Rb family protein, is significantly reduced in both ERK2-shRNA cells upon IR and ERK1/2-shRNA cells without IR. ERK1- or ERK1/2-shRNA cells failed to recover G2-M phase while ERK2 shRNA cells progress into G2-M phase as well as control cells. Our study indicates that PCa cells that survive high-dose IR display an aggressive phenotype characterized by constitutively up-regulation of ERK1/2 activity and plays an important role in cell survival following radiation. Although down-regulation of ERK1 and/or ERK2 can sensitize cells to IR, the mechanisms influencing cell survival may be different. Upon IR, ERK1 appears to influence BCl2 family and regulation of the cell cycle. ERK2 has impact on XIAP function and has less effect on the cell cycle. These findings define a mechanism of cellular resistance to IR therapy and they suggest that strategies aimed to limit the effects of ERK signaling pathway could be beneficial and influence multiple targets.

Abstract LB-080: Reactivating RBL2/p130 oncosuppressive function as a new, possible antitumoral strategy

Abstract Deregulation of cell cycle control is the leading cause of cancer. The retinoblastoma (Rb) family members, including RB1/p105, RBL1/p107 and RBL2/p130, are crucial to restrain cell cycle progression and their inactivation, either direct or indirect, is a hallmark of most human tumors. In particular, RBL2/p130 emerging role in senescence and apoptosis, seems to contribute importantly to its tumor suppressor function. Furthermore, many studies largely contributed to establish RBL2/p130 as an important cancer target, which is inactivated by cell cycle kinases and whose deregulation underlies various cancer types. In this regard, we set out to restore RBL2/p130 function in tumors and exploit its tumor suppressive potential for cancer therapy. In particular, we have identified, through computational chemistry and molecular modeling studies, a small molecule able to act as a specific inhibitor of the CDK2-CycA complex and to reactivate the tumor suppressive function of RBL2/p130 in cancer. We analyzed by MTS assay the cytotoxic effect of our compound on a panel of different tumor cell lines and determined its IC50 values. We evaluated its effects on cell cycle and apoptosis by FACS and dissected its molecular mechanism of action by Western blot and RT-PCR. We found that our compound effectively inhibited proliferation of lung cancer and mesothelioma cell lines by specifically inhibiting CDK2-CycA activity towards RBL2/p130. The decrease in RBL2/p130 phosphorylation led to stabilization of its complex with the E2F4 transcriptional factor and consequent repression of their targets necessary for cell cycle progression, including CDK2 itself. Consistently, our compound arrested cell cycle and triggered apoptosis. Interestingly, apoptosis seemed to be mediated by RBL2/p130 itself because it was repressed in RBL2/p130-silenced cells. Furthermore, our compound proved to be active in an A549 xenograft model of lung cancer. Overall our findings indicate that the pharmacological reactivation of RBL2/p130 induces its cell-cycle restraining and pro-apoptotic functions, the latter being still largely unexplored, and identify a new possible therapeutic strategy for cancer treatment. Citation Format: Francesca Pentimalli, Luca Esposito, Iris Maria Forte, Carmelina Antonella Iannuzzi, Flavio Rizzolio, Tiziano Tuccinardi, Paola Indovina, Silvia Boffo, Antonio Giordano. Reactivating RBL2/p130 oncosuppressive function as a new, possible antitumoral strategy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-080. doi:10.1158/1538-7445.AM2015-LB-080