Project CFTREMT Supervisor Margarida Amaral FCULBio - PDF document

1 Project CFTR‐EMT ‐ Supervisor: Marga
Project CFTR‐EMT ‐ Supervisor: Margarida Amaral (FCUL‐Bio ISI), Co‐supervisor: Jonas Fuxe (Karolinska Institutet) Title: Role of CFTR in epithelial mesenchymal transition (EMT) by functional genomics Objectives: To identify CFTR ‐ related epithelial differentiation pathways by functional genomics Methodology: Cystic Fibrosis (CF), the most comm on life shortenin g, genetic disease in Europe is caused by mutations in CFTR protein, a chloride channel expressed at apical membrane of epithelia and a master regulator of epithelial ion and water homeostasis͘ The most frequent CF‐ causing mutation, F508de l – leads to protein misfolding, a trafficking defect due to retention at the endoplasmic reticulum (ER) and premature proteasomal degradatio n. An emerging body of evidence suggests that CFTR also plays a rol e in epithelial cell differentiation. T his includes: 1) a delay in the epithelial differentiation process in CF vs non‐CF cells [1]͖ 2) functional CFTR is required for the rapid regeneration of human airway surface epithelium after injury [2]; 3) a signifi cant set of differentiation genes are differentially expressed in CF vs non‐CF tissue s [3] ; 4) loss of CFTR leads to abnormal tracheal development in neonatal mice, pigs and children [4]; 5) CFTR downregulation promotes epithelial‐to‐ mesenchymal transition (EMT) and correlate s with poor prognosis of cancer [5,6]; 6) CF patients have an increased risk of digestive tract and other cancer forms [7]. Our hypothesis is that disruption of the airway epithelial diff erentiation due to absence of functional CFTR is a central defect in CF, and that in turn a network of epit helial‐specific factors affects secretory traffic of CFTR and other proteins that are characteristic of highly differentiated epithelia. In ongoing wo rk in the host lab, using microscopy based genome ‐ scal e siRNA screens we use a robust cell ‐ based assay for monitoring the effects of siRNAs on CFTR plasm a membrane traffic [8]. So far, knock ‐ down of ~180 genes enhanced CFTR plasma membrane traffic (Amaral lab, unpublished data). Moreover, by applying a similar assay to the most frequent F508del ‐ CFTR mut ant, we also found a set of genes which when down ‐ regulated rescue this mutant to the ce ll surface. Some of these genes are implicated in epithelial cell dif ferentiation. In another global gen omic approach identifying genes differentially expressed in CF vs non ‐ CF human native respiratory epit helial cells, we found a set of genes related epithelial cell differentiation among the most signific antly disturbed in CF [9]. Here, we will determine by high ‐ content microscopy screens which of t hose previously identified hits from CFTR screen which are differentiation ‐ related also play a role in EMT. To this end we will re ‐ test the effec

2 ts of their siRNAs under condition s tha
ts of their siRNAs under condition s that affect EMT such as knock ‐ down of CEBPß or miR155 [10]. The data will be used in the construction of gene regulatory net works shared by EMT and traffic pathways. Understanding how F508del ‐ CFTR is rescued to the cell surface by this network will enabl e us to identify novel drug targets for CF and mimic such effects by small molecules differentially expressed in CF vs non ‐ CF human native respiratory epit heli al cells, we found a set of genes related epithelial cell differentiation among the most significantly disturbed in CF [9]. [1] Hajj R et al (2007). Human airway surface epithelial regeneration is delayed and abnormal in cystic fibrosis. J Pathol 211 : 340‐350͘ [2] Puchelle E et al (2006) Airway epithelial repair, regeneration, and remodeling afte r injury in chronic obstructive pulmonary disease. Proc Am Thorac Soc 3 : 726‐733͘ [3] Clarke LA et al (2013) Changes in transcriptome of native nasal epithelium expressin g F508del‐CFTR and intersecting data from comparable studies. Respir Res 14 : 38. [4] Bonvin E et al (2008) Congenital tracheal malformation in cystic fibrosis transmembrane conductance regulatordeficient mice. J Physiol 586 : 3231‐3243͖ Meyerholz DK et al (2010) Loss of cystic fibrosis tr ansmembrane conductance regulator function produces abnormalities in tracheal development in ne onatal pigs and young children. Am J Respir Crit Care Med 182 : 1251‐1261͘ [5] Zhang JT et al (2013) Downregulation of CFTR promotes epithelial‐to‐mesenchymal tra nsition and is associated with poor prognosis of breast cancer. Biochim Biophys Acta 1833 : 2961‐2969 [6] Xie C et al (2013) CFTR suppresses tumor progression through miR‐193b targeting urokinase plasminogen activator (uPA) in prostate cancer. Oncogene 32 : 2282‐91, 2291͘ [7] Maisonneuve P et al (2013) Cancer risk in cystic fibrosis: a 20‐year nationwide study from the United States͘ J Natl Cancer Inst 105 : 122‐129 [8] Botelho et al (2015) Protein Traffic Disorders: an Effective :igh‐Throughput Fluorescence Mi croscopy Pipeline for Drug Discovery. Sci Rep 5 : 9038. [9] Clarke LA et al (2013) Transcriptomic changes in native nasal epithelium expressing F508del‐ CFTR and contrasting data from comparable studies. Resp Res 14 : 38. [10] Johansson J et al (2013) MiR‐155 ‐mediated loss of C/EBP β shifts the TGF‐ß res ponse from growth inhibition to epithelial mesenchymal transition, invasion and metastasis in breast cancer. Oncogene 32 : 5614‐24͘ Supervisor : Margarida D. Amaral, BioISI /FCUL Co ‐ Supervisor: Jonas Fuxe, Karolinsk a Institutet, Sweden Type of fellowship : Mixed (Portugal and abroad): FCUL/BioISI and Karolinska Institutet , Stockholm