DOCSLIB.ORG

Regulation of Mitochondrial Function by Myocardin During Cardiac Development and Disease

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Regulation of Mitochondrial Function by Myocardin During Cardiac Development and Disease Regulation of mitochondrial function by Myocardin during cardiac development and disease By Wajihah Mughal A Thesis submitted to the Faculty of Graduate Studies University of Manitoba In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Human Anatomy and Cell Science University of Manitoba Winnipeg Copyright © by Wajihah Mughal, 2019 Abstract Metabolic specific myogenic precursors in the splanchnic, somatic mesoderm, and the neural crest give rise to specialized cell types such as cardiac, skeletal, and smooth muscle cells. This initiation of muscle cell lineage is coordinated by a reinforcing networking of transcription factors that regulate gene expression during cell proliferation and differentiation. Myocardin is a transcriptional coactivator that binds to transcription factors to regulate gene expression specific to both cardiac and smooth muscle cells. It is previously shown that Myocardin interacts with transcription factors of the MADS-Box family proteins such as myocyte enhancer factor-2 (MEF2) and serum response factor (SRF), that are known regulators of cellular differentiation and metabolism. Conversely, the role of Myocardin as well as its regulation of MADS-Box transcription factors and mitochondrial function during development and disease is not well understood. Therefore, we chose to investigate a Myocardin-regulated genetic pathway that regulates mitochondrial function in cardiac muscle that becomes dysregulated during disease. My thesis summarizes our evaluation of the hypothesis in two studies. In the first study we characterize a mechanistic pathway involving MEF2 and SRF, that regulates mitochondrial function in all three muscle lineages. This initial study demonstrates a genetic pathway in which Nix is a direct target of miR-133a, its role in regulating insulin sensitivity and metabolic dysfunction in myocytes. These novel findings lead to my second study that examines the role of Myocardin in regulating miR-133a and Nix in cardiac cells. I provide evidence that miR−133a is a direct transcriptional target of Myocardin that regulates mitochondrial permeability transition to preserve cell survival during cardiac development and following myocardial ischemic injury. ii Together, these findings inform researchers about the vital role of a Myocardin- regulated pathway that maintains mitochondrial function to oppose cell death during development as novel pharmacotherapy targets. Ongoing investigation is required to further understand miR-133a function as a potential cardiometabolic biomarker or as a therapeutic intervention for the prevention and/or treatment of congenital heart defects, cardiometabolic disease and heart failure. iii Dedication This thesis is dedicated to my parents, Dost and Rehana Mughal, to my siblings and loved ones for their continuous love, encouragement, support and guidance, whose sacrifices made it possible for me to complete this work, and without whom none of my success would be possible. to my chand, Jason Van Zwol for being an incredible husband, for being the rock in my life, for always encouraging me to be myself, for supporting me emotionally and mentally, and for loving me unconditionally since the day we met. to my supervisor Dr Joe Gordon and mentors for supporting my research goals and enthusiasm for science to my fellow trainees for their friendship that will last beyond grad school iv and lastly, this thesis is dedicated to the women in science we all move forward when we recognize how resilient and striking the women around us are - rupi kaur v Acknowledgements I would like to express my deepest gratitude and sincerest appreciation to my doctoral supervisor, mentor and career advisor, Dr Joseph Gordon. His limitless support, guidance, motivation, and friendship has greatly inspired me throughout my journey as a grad student. However, his mentorship started four years earlier during my masters studies while he was completing his post-doctoral fellowship. Early on, I saw many qualities in him that would make him an outstanding investigator, not knowing that I would be become his trainee one day. It is an honour that he took me under his wing, saw potential in me and had confidence in my skills as his first PhD student. Joe’s outstanding supervision, leadership, honesty, research enthusiasm, down to earth and caring personality, combined with our shared amusement for corny science jokes (“it Hoechst don’t it?”) has made this difficult rollercoaster of a journey a fun and unforgettable one. I will be forever appreciative and thankful for his shared wisdom and knowledge of literally everything and for supporting my research directions. I am thankful for how he would challenge and push me beyond my boundaries to think critically. I am thankful that he saw my potential as a cell biologist that has allowed me to advance my technical skillset, research knowledge, writing and presentation style under his supervision. My supervisor has inspired me to pursue my goals, work with dedication and determination no matter what hurdles I had to face. His confidence in me has allowed me to persevere through many challenges that has led to my personal growth and professional success. Under his supervision and mentorship, my research was provincially funded, published in prestigious journals, presented at multiple international conferences, which has allowed me vi to complete my thesis within a timely manner. My supervisor has always allowed for my career growth which is best demonstrated by his full support when I was applying for my Mitacs position and how he helped me prep for my interview. Joe has taught me some incredible life lessons that I am forever grateful for: he has always emphasized the importance of balancing work and personal life, to recharge when needed, and encourage creativity. While being my advisor and good friend, I can strongly assert that each and every supervisor should have qualities like Joe; he is the most outstanding mentor any student at any academic level could ask for. The completion of this work would not have been possible without his continuous support and guidance. I would like to extend my gratitude to my doctoral advisory committee members, Dr Thomas Klonish, Dr Ian Dixon, and Dr Saeid Ghavami, for their constant encouragement and support throughout my studies. I am also thankful to my external examiner, Dr Lynn Megeney, for his time in travelling to attend my defense and offer his valuable insight to my research project. It was an honour and a memorable experience to defend my thesis to such an esteemed committee panel. The studies presented in this document were supported by many collaborators that made it possible to publish our research whom I am very thankful for. This includes Dr Michael Parmacek from the University of Pennsylvania, Dr Ian Dixon from St. Boniface Research Centre, and our multiple collaborators from the Children’s Hospital Research Institute including Dr William Diehl-Jones, Dr Vern Dolinsky, Dr Richard Keijzer, Dr Grant Hatch, and Dr Adrian West. vii I would also like to thank Research Manitoba (formerly MHRC), the Children’s Hospital Research Institute (CHRIM), the University of Manitoba’s Faculty of Graduate Studies, and the Diabetes Research Envisioned & Accomplished in Manitoba (DREAM), for financially supporting this research project. I was also fortunate to receive many poster, travel, and research awards from various funding organizations for which I am very grateful for. For the past and present members of the Department of Human Anatomy and Cell Science administrative staff, including Jennifer Genest, Jacki Armstrong, and Martha Ericastilla, I am appreciative of their support and for keeping things fun between seminars/deadlines. I am forever grateful for the healthy and supportive lab environment that has had a very positive impact on my quality of research, success and productivity. I am fortunate to belong to a lab family that has gone above and beyond in supporting this work. I greatly appreciate our easy-going and exceptionally skilled technician, Donald Chapman, for his friendship and for being an integral part of the research presented in this thesis. I appreciate the days we spent in the cold room crushing tissues and I still remember our first time isolating neonatal hearts together (we are heart breakers). I appreciate all the times I spent with a good friend and fomer technician, Dr Yan Hai, for her kind heart and patience in teaching me highly difficult skills; whose presence and positivity brightened up the lab every day. I am grateful for my lab mates, Matthew Martens and Jared Field, for their friendship, and random lab banter that always kept things interesting in the lab. I am grateful for the time we spent at our first conference in San Diego at EB, all the work they put into getting the viii Myocardin manuscript out, and how they spent time awat from their research to help me prepare for my dissertation. We didn’t get a long at first, but I am grateful for their support, friendship, and for making the lab such an enjoyable environment to learn in. I am thankful to Simone da Silva Rosa for her work ethic, her enthusiasm for science and for being among my first summer students in the lab. I am also appreciative of many past members of the Gordon lab including Adrian Ancheta, Alyssa Archibald, Lucas Nguyen, Steven Piotrowski, Tayna Fiuza, and Adel Rezai Moghadam, for being wonderful colleagues and their contributions towards this work. I am extremely appreciative of Stephanie Kereliuk, my PhD soul sister, for her unconditional friendship, guidance, and continuous support. Every grad student should have a friend like her; she has always encouraged me to do my best, and always reminded me that I could do anything, but I just had to be myself. There aren’t enough words to describe how she has been such an important and exceptionally positive part of my grad school experience, and I know our friendship will last for many years ahead.
Load more

Recommended publications
  • MKL1 (C) Antibody, Rabbit Polyclonal
    Order: (888)-282-5810 (Phone) (818)-707-0392 (Fax) [email protected] Web: www.Abiocode.com MKL1 (C) Antibody, Rabbit Polyclonal Cat#: R2403-2 Lot#: Refer to vial Quantity: 100 ul Application: WB Predicted I Observed M.W.: 99 I 140 kDa Uniprot ID: Q969V6 Background: MKL/myocardin-like protein 1 (MKL1) is a transcriptional coactivator of serum response factor (SRF) with the potential to modulate SRF target genes. MKL1 suppresses TNF-induced cell death by inhibiting activation of caspases; its transcriptional activity is indispensable for the antiapoptotic function. MKL1 may up-regulate antiapoptotic molecules, which in turn inhibit caspase activation. Other Names: MKL/myocardin-like protein 1, Megakaryoblastic leukemia 1 protein, Megakaryocytic acute leukemia protein, Myocardin-related transcription factor A, MRTF-A, KIAA1438, MAL Source and Purity: Rabbit polyclonal antibodies were produced by immunizing animals with a GST-fusion protein containing the C-terminal region of human MKL1. Antibodies were purified by affinity purification using immunogen. Storage Buffer and Condition: Supplied in 1 x PBS (pH 7.4), 100 ug/ml BSA, 40% Glycerol, 0.01% NaN3. Store at -20 °C. Stable for 6 months from date of receipt. Species Specificity: Human Tested Applications: WB: 1:1,000-1:3,000 (detect endogenous protein*) *: The apparent protein size on WB may be different from the calculated M.W. due to modifications. For research use only. Not for therapeutic or diagnostic purposes. Abiocode, Inc., 29397 Agoura Rd., Ste 106, Agoura Hills, CA 91301 Order: (888)-282-5810 (Phone) (818)-707-0392 (Fax) [email protected] Web: www.Abiocode.com Product Data: kDa A B 250 150 100 75 50 37 Fig 1.
    [Show full text]
  • Wide-Scale Analysis of Human Functional Transcription Factor
    Wide-Scale Analysis of Human Functional Transcription Factor Binding Reveals a Strong Bias towards the Transcription Start Site Yuval Tabach1,2, Ran Brosh2, Yossi Buganim2, Anat Reiner1, Or Zuk1, Assif Yitzhaky1, Mark Koudritsky1, Varda Rotter2 and Eytan Domany1,* 1Departments of Physics of Complex Systems and 2Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel *Corresponding author. Email: [email protected] Tel: +972-8-9343964 Fax: +972-8- 9344109 Running title: Binding Sites Location Bias Keywords: Transcription factor binding sites, location bias, cell cycle, NF-kB, promoter, Gene Ontology, TATA, myocardin 1 Summary Background: Elucidating basic principles that underlie regulation of gene expression by transcription factors (TFs) is a central challenge of the post-genomic era. Transcription factors regulate expression by binding to specific DNA sequences; such a binding event is functional when it affects gene expression. Functionality of a binding site is reflected in conservation of the binding sequence during evolution and in over represented binding in gene groups with coherent biological functions. Functionality is governed by several parameters such as the TF- DNA binding strength, distance of the binding site from the transcription start site (TSS), DNA packing, and more. Understanding how these parameters control functionality of different TFs in different biological contexts is an essential step towards identifying functional TF binding sites, a must for understanding regulation of transcription. Methodology/Principal Findings: We introduce a novel method to screen the promoters of a set of genes with shared biological function, against a precompiled library of motifs, and find those motifs which are statistically over-represented in the gene set.
    [Show full text]
  • Co-Occupancy by Multiple Cardiac Transcription Factors Identifies
    Co-occupancy by multiple cardiac transcription factors identifies transcriptional enhancers active in heart Aibin Hea,b,1, Sek Won Konga,b,c,1, Qing Maa,b, and William T. Pua,b,2 aDepartment of Cardiology and cChildren’s Hospital Informatics Program, Children’s Hospital Boston, Boston, MA 02115; and bHarvard Stem Cell Institute, Harvard University, Cambridge, MA 02138 Edited by Eric N. Olson, University of Texas Southwestern, Dallas, TX, and approved February 23, 2011 (received for review November 12, 2010) Identification of genomic regions that control tissue-specific gene study of a handful of model genes (e.g., refs. 7–10), it has not been expression is currently problematic. ChIP and high-throughput se- evaluated using unbiased, genome-wide approaches. quencing (ChIP-seq) of enhancer-associated proteins such as p300 In this study, we used a modified ChIP-seq approach to define identifies some but not all enhancers active in a tissue. Here we genome wide the binding sites of these cardiac TFs (1). We show that co-occupancy of a chromatin region by multiple tran- provide unbiased support for collaborative TF interactions in scription factors (TFs) identifies a distinct set of enhancers. GATA- driving cardiac gene expression and use this principle to show that chromatin co-occupancy by multiple TFs identifies enhancers binding protein 4 (GATA4), NK2 transcription factor-related, lo- with cardiac activity in vivo. The majority of these multiple TF- cus 5 (NKX2-5), T-box 5 (TBX5), serum response factor (SRF), and “ binding loci (MTL) enhancers were distinct from p300-bound myocyte-enhancer factor 2A (MEF2A), here referred to as cardiac enhancers in location and functional properties.
    [Show full text]
  • Myocardin (MYOCD) (NM 001146312) Human Tagged ORF Clone Product Data
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for RC229055L3 Myocardin (MYOCD) (NM_001146312) Human Tagged ORF Clone Product data: Product Type: Expression Plasmids Product Name: Myocardin (MYOCD) (NM_001146312) Human Tagged ORF Clone Tag: Myc-DDK Symbol: MYOCD Synonyms: MGBL; MYCD Vector: pLenti-C-Myc-DDK-P2A-Puro (PS100092) E. coli Selection: Chloramphenicol (34 ug/mL) Cell Selection: Puromycin ORF Nucleotide The ORF insert of this clone is exactly the same as(RC229055). Sequence: Restriction Sites: SgfI-MluI Cloning Scheme: ACCN: NM_001146312 ORF Size: 2958 bp This product is to be used for laboratory only. Not for diagnostic or therapeutic use. View online » ©2021 OriGene Technologies, Inc., 9620 Medical Center Drive, Ste 200, Rockville, MD 20850, US 1 / 3 Myocardin (MYOCD) (NM_001146312) Human Tagged ORF Clone – RC229055L3 OTI Disclaimer: Due to the inherent nature of this plasmid, standard methods to replicate additional amounts of DNA in E. coli are highly likely to result in mutations and/or rearrangements. Therefore, OriGene does not guarantee the capability to replicate this plasmid DNA. Additional amounts of DNA can be purchased from OriGene with batch-specific, full-sequence verification at a reduced cost. Please contact our customer care team at [email protected] or by calling 301.340.3188 option 3 for pricing and delivery. The molecular sequence of this clone aligns with the gene accession number as a point of reference only. However, individual transcript sequences of the same gene can differ through naturally occurring variations (e.g.
    [Show full text]
  • Gene Expression During Normal and FSHD Myogenesis Tsumagari Et Al
    Gene expression during normal and FSHD myogenesis Tsumagari et al. Tsumagari et al. BMC Medical Genomics 2011, 4:67 http://www.biomedcentral.com/1755-8794/4/67 (27 September 2011) Tsumagari et al. BMC Medical Genomics 2011, 4:67 http://www.biomedcentral.com/1755-8794/4/67 RESEARCHARTICLE Open Access Gene expression during normal and FSHD myogenesis Koji Tsumagari1, Shao-Chi Chang1, Michelle Lacey2,3, Carl Baribault2,3, Sridar V Chittur4, Janet Sowden5, Rabi Tawil5, Gregory E Crawford6 and Melanie Ehrlich1,3* Abstract Background: Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35. Within each repeat unit is a gene, DUX4, that can encode a protein containing two homeodomains. A DUX4 transcript derived from the last repeat unit in a contracted array is associated with pathogenesis but it is unclear how. Methods: Using exon-based microarrays, the expression profiles of myogenic precursor cells were determined. Both undifferentiated myoblasts and myoblasts differentiated to myotubes derived from FSHD patients and controls were studied after immunocytochemical verification of the quality of the cultures. To further our understanding of FSHD and normal myogenesis, the expression profiles obtained were compared to those of 19 non-muscle cell types analyzed by identical methods. Results: Many of the ~17,000 examined genes were differentially expressed (> 2-fold, p < 0.01) in control myoblasts or myotubes vs. non-muscle cells (2185 and 3006, respectively) or in FSHD vs. control myoblasts or myotubes (295 and 797, respectively). Surprisingly, despite the morphologically normal differentiation of FSHD myoblasts to myotubes, most of the disease-related dysregulation was seen as dampening of normal myogenesis- specific expression changes, including in genes for muscle structure, mitochondrial function, stress responses, and signal transduction.
    [Show full text]
  • Myocardin Is Sufficient and Necessary for Cardiac Gene Expression in Xenopus Eric M
    Research article 987 Myocardin is sufficient and necessary for cardiac gene expression in Xenopus Eric M. Small1,*,†, Andrew S. Warkman1,*, Da-Zhi Wang2, Lillian B. Sutherland3, Eric N. Olson3 and Paul A. Krieg1,‡ 1Department of Cell Biology and Anatomy, University of Arizona Health Sciences Center, 1501 N. Campbell Avenue, PO Box 245044, Tucson, AZ, 85724, USA 2Carolina Cardiovascular Biology Center, Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC 27599-7126, USA 3Department of Molecular Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9148, USA *These authors contributed equally to this work †Present address: Cardiovascular Research, The Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada ‡Author for correspondence (e-mail: [email protected]) Accepted 14 December 2004 Development 132, 987-997 Published by The Company of Biologists 2005 doi:10.1242/dev.01684 Summary Myocardin is a cardiac- and smooth muscle-specific co- expression. Inhibition of myocardin activity in Xenopus factor for the ubiquitous transcription factor serum embryos using morpholino knockdown methods results in response factor (SRF). Using gain-of-function approaches inhibition of cardiac development and the absence of in the Xenopus embryo, we show that myocardin is expression of cardiac differentiation markers and severe sufficient to activate transcription of a wide range of disruption of cardiac morphological processes. We cardiac and
    [Show full text]
  • Loss-Of-Function Variants in Myocardin Cause Congenital Megabladder in Humans and Mice
    Loss-of-function variants in myocardin cause congenital megabladder in humans and mice Arjan C. Houweling, … , Adrian S. Woolf, Esther E. Creemers J Clin Invest. 2019. https://doi.org/10.1172/JCI128545. Concise Communication In-Press Preview Muscle biology Myocardin (MYOCD) is the founding member of a class of transcriptional co-activators that bind serum response factor to activate gene expression programs critical in smooth muscle (SM) and cardiac muscle development. Insights into the molecular functions of MYOCD have been obtained from cell culture studies and, to date, knowledge about in vivo roles of MYOCD comes exclusively from experimental animals. Here, we defined an often lethal congenital human disease associated with inheritance of pathogenic MYOCD variants. This disease manifested as a massively dilated urinary bladder, or megabladder, with disrupted SM in its wall. We provided evidence that monoallelic loss-of-function variants in MYOCD caused congenital megabladder in males only, whereas biallelic variants were associated with disease in both sexes, with a phenotype additionally involving the cardiovascular system. These results were supported by co- segregation of MYOCD variants with the phenotype in four unrelated families, by in vitro transactivation studies where pathogenic variants resulted in abrogated SM gene expression, and finding megabladder in two distinct mouse models with reduced Myocd activity. In conclusion, we have demonstrated that variants in MYOCD result in human disease, and the collective findings highlight a vital role for MYOCD in mammalian organogenesis. Find the latest version: https://jci.me/128545/pdf Loss-of-function variants in myocardin cause congenital megabladder in humans and mice Arjan C.
    [Show full text]
  • The Myocardin Family of Transcriptional Coactivators: Versatile Regulators of Cell Growth, Migration, and Myogenesis
    Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration, and myogenesis G.C. Teg Pipes, Esther E. Creemers, and Eric N. Olson1 Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA The association of transcriptional coactivators with se- gene regulation and expands the regulatory potential of quence-specific DNA-binding proteins provides versatil- individual cis-regulatory elements. ity and specificity to gene regulation and expands the The regulation of serum response factor (SRF) and its regulatory potential of individual cis-regulatory DNA se- target genes provides a classic example of the diversity of quences. Members of the myocardin family of coactiva- genes controlled by a single DNA-binding protein and tors activate genes involved in cell proliferation, migra- the importance of cofactor interactions in the control of tion, and myogenesis by associating with serum re- gene expression (Shore and Sharrocks 1995). Among the sponse factor (SRF). The partnership of myocardin family many target genes of SRF are genes involved in cell pro- members and SRF also controls genes encoding compo- liferation and muscle differentiation, which represent nents of the actin cytoskeleton and confers responsive- opposing programs of gene expression: Muscle genes are ness to extracellular growth signals and intracellular repressed by growth factor signals and generally do not changes in the cytoskeleton, thereby creating a tran- become activated until myoblasts exit the cell cycle. The scriptional–cytoskeletal regulatory circuit. These func- ability of SRF to regulate different sets of downstream tions are reflected in defects in smooth muscle differen- effector genes depends on its association with positive tiation and function in mice with mutations in myocar- and negative cofactors (Treisman 1994; Shore and Shar- din family members.
    [Show full text]
  • Signaling and Transcriptional Networks in Heart Development and Regeneration
    Downloaded from http://cshperspectives.cshlp.org/ on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Signaling and Transcriptional Networks in Heart Development and Regeneration Benoit G. Bruneau Gladstone Institute of Cardiovascular Disease, San Francisco, California 94158, and Department of Pediatrics and Cardiovascular Research Institute, University of California, San Francisco, California 94158 Correspondence: [email protected] SUMMARY The mammalian heart is the first functional organ, the first indicator of life. Its normal formation and function are essential for fetal life. Defects in heart formation lead to congenital heart defects, underscoring the finesse with which the heart is assembled. Understanding the reg- ulatory networks controlling heart development have led to significant insights into its lineage origins and morphogenesis and illuminated important aspects of mammalian embryology, while providing insights into human congenital heart disease. The mammalian heart has very little regenerative potential, and thus, any damage to the heart is life threatening and perma- nent. Knowledge of the developing heart is important for effective strategies of cardiac regen- eration, providing new hope for future treatments for heart disease. Although we still have an incomplete picture of the mechanisms controlling development of the mammalian heart, our current knowledge has important implications for embryology and better understanding of human heart disease. Outline 1 Introduction 4 Heart repair and
    [Show full text]
  • Inhibition of the Myocardin-Related Transcription Factor Pathway Increases Efficacy of Trametinib in NRAS-Mutant Melanoma Cell L
    cancers Article Inhibition of the Myocardin-Related Transcription Factor Pathway Increases Efficacy of Trametinib in NRAS-Mutant Melanoma Cell Lines Kathryn M. Appleton 1, Charuta C. Palsuledesai 1, Sean A. Misek 2, Maja Blake 1, Joseph Zagorski 1, Kathleen A. Gallo 2, Thomas S. Dexheimer 1 and Richard R. Neubig 1,3,* 1 Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824, USA; [email protected] (K.M.A.); [email protected] (C.C.P.); [email protected] (M.B.); [email protected] (J.Z.); [email protected] (T.S.D.) 2 Department of Physiology, Michigan State University, East Lansing, MI 48824, USA; [email protected] (S.A.M.); [email protected] (K.A.G.) 3 Department of Medicine, Division of Dermatology, Michigan State University, East Lansing, MI 48824, USA * Correspondence: [email protected]; Tel.: +1-517-353-7145 Simple Summary: Malignant melanoma is the most aggressive skin cancer, and treatment is often ineffective due to the development of resistance to targeted therapeutic agents. The most prevalent form of melanoma with a mutated BRAF gene has an effective treatment, but the second most common mutation in melanoma (NRAS) leads to tumors that lack targeted therapies. In this study, we show that NRAS mutant human melanoma cells that are most resistant to inhibition of the oncogenic pathway have a second activated pathway (Rho). Inhibiting that pathway at one of several Citation: Appleton, K.M.; points can produce more effective cell killing than inhibition of the NRAS pathway alone. This raises Palsuledesai, C.C.; Misek, S.A.; Blake, the possibility that such a combination treatment could prove effective in those melanomas that fail M.; Zagorski, J.; Gallo, K.A.; to respond to existing targeted therapies such as vemurafenib and trametinib.
    [Show full text]
  • Genome-Wide RNA-Sequencing Analysis Identifies a Distinct
    www.nature.com/scientificreports OPEN Genome-wide RNA-Sequencing analysis identifies a distinct fibrosis gene signature in the conjunctiva Received: 16 March 2017 Accepted: 27 June 2017 after glaucoma surgery Published: xx xx xxxx Cynthia Yu-Wai-Man1,2, Nicholas Owen2, Jonathan Lees3, Aristides D. Tagalakis4, Stephen L. Hart 4, Andrew R. Webster1,2, Christine A. Orengo3 & Peng T. Khaw1,2 Fibrosis-related events play a part in most blinding diseases worldwide. However, little is known about the mechanisms driving this complex multifactorial disease. Here we have carried out the first genome-wide RNA-Sequencing study in human conjunctival fibrosis. We isolated 10 primary fibrotic and 7 non-fibrotic conjunctival fibroblast cell lines from patients with and without previous glaucoma surgery, respectively. The patients were matched for ethnicity and age. We identified 246 genes that were differentially expressed by over two-fold andp < 0.05, of which 46 genes were upregulated and 200 genes were downregulated in the fibrotic cell lines compared to the non-fibrotic cell lines. We also carried out detailed gene ontology, KEGG, disease association, pathway commons, WikiPathways and protein network analyses, and identified distinct pathways linked to smooth muscle contraction, inflammatory cytokines, immune mediators, extracellular matrix proteins and oncogene expression. We further validated 11 genes that were highly upregulated or downregulated using real-time quantitative PCR and found a strong correlation between the RNA-Seq and qPCR results. Our study demonstrates that there is a distinct fibrosis gene signature in the conjunctiva after glaucoma surgery and provides new insights into the mechanistic pathways driving the complex fibrotic process in the eye and other tissues.
    [Show full text]
  • MRTFB Suppresses Colorectal Cancer Development Through Regulating SPDL1 and MCAM
    MRTFB suppresses colorectal cancer development through regulating SPDL1 and MCAM Takahiro Kodamaa,b,c, Teresa A. Mariana,b, Hubert Leea, Michiko Kodamaa, Jian Lid, Michael S. Parmacekd, Nancy A. Jenkinsa,e, Neal G. Copelanda,e,1, and Zhubo Weia,b,1 aHouston Methodist Research Institute, Houston Methodist Hospital, Houston, TX 77030; bHouston Methodist Cancer Center, Houston Methodist Hospital, Houston, TX 77030; cDepartment of Gastroenterology and Hepatology, Graduate School of Medicine, Osaka University, 5650871 Suita, Osaka, Japan; dDepartment of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104; and eGenetics Department, The University of Texas MD Anderson Cancer Center, Houston, TX 77030 Contributed by Neal G. Copeland, October 7, 2019 (sent for review June 18, 2019; reviewed by Masaki Mori and Hiroshi Seno) Myocardin-related transcription factor B (MRTFB) is a candidate tumor- shown to regulate cell cycle progression (9) and HCC xenograft suppressor gene identified in transposon mutagenesis screens of the tumor growth (10). Functional validation using cell culture sys- intestine, liver, and pancreas. Using a combination of cell-based assays, tems have also shown that reduced expression of MRTFB by in vivo tumor xenograft assays, and Mrtfb knockout mice, we demon- RNA interference leads to increased CRC cell invasion (4), strate here that MRTFB is a human and mouse colorectal cancer suggesting its important role in tumor progression. (CRC) tumor suppressor that functions in part by inhibiting cell Based on these results, we decided to conditionally delete invasion and migration. To identify possible MRTFB transcriptional Mrtfb in the mouse intestine to further explore its role in CRC.
    [Show full text]