title: Genetic disruption of the pHi-regulating proteins Na+/H+ exchanger 1 (SLC9A1) and carbonic anhydrase 9 severely reduces growth of colon cancer cells.
abstract: Hypoxia and extracellular acidosis are pathophysiological hallmarks of aggressive solid tumors. Regulation of intracellular pH (pHi) is essential for the maintenance of tumor cell metabolism and proliferation in this microenvironment and key proteins involved in pHi regulation are of interest for therapeutic development. Carbonic anhydrase 9 (CA9) is one of the most robustly regulated proteins by the hypoxia inducible factor (HIF) and contributes to pHi regulation. Here, we have investigated for the first time, the role of CA9 via complete genomic knockout (ko) and compared its impact on tumor cell physiology with the essential pHi regulator Na+/H+ exchanger 1 (NHE1). Initially, we established NHE1-ko LS174 cells with inducible CA9 knockdown. While increased sensitivity to acidosis for cell survival in 2-dimensions was not observed, clonogenic proliferation and 3-dimensional spheroid growth in particular were greatly reduced. To avoid potential confounding variables with use of tetracycline-inducible CA9 knockdown, we established CA9-ko and NHE1/CA9-dko cells. NHE1-ko abolished recovery from NH4Cl pre-pulse cellular acid loading while both NHE1 and CA9 knockout reduced resting pHi. NHE1-ko significantly reduced tumor cell proliferation both in normoxia and hypoxia while CA9-ko dramatically reduced growth in hypoxic conditions. Tumor xenografts revealed substantial reductions in tumor growth for both NHE1-ko and CA9-ko. A notable induction of CA12 occurred in NHE1/CA9-dko tumors indicating a potential means to compensate for loss of pH regulating proteins to maintain growth. Overall, these genomic knockout results strengthen the pursuit of targeting tumor cell pH regulation as an effective anti-cancer strategy.
authors.affiliation: Medical Biology Department, Centre Scientifique de Monaco (CSM), Monaco.
authors.forename: Scott K
authors.initials: SK
authors.lastname: Parks
authors.affiliation: Medical Biology Department, Centre Scientifique de Monaco (CSM), Monaco.
authors.forename: Yann
authors.initials: Y
authors.lastname: Cormerais
authors.affiliation: Medical Biology Department, Centre Scientifique de Monaco (CSM), Monaco.
authors.forename: Jerome
authors.initials: J
authors.lastname: Durivault
authors.affiliation: Medical Biology Department, Centre Scientifique de Monaco (CSM), Monaco.|Institute for Research on Cancer & Aging (IRCAN), CNRS, INSERM, Centre A. Lacassagne, University of Nice-Sophia Antipolis, Nice, France.
authors.forename: Jacques
authors.initials: J
authors.lastname: Pouyssegur
mesh_terms: D000230:Adenocarcinoma / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics* / Q000378:metabolism; D064113:CRISPR-Cas Systems; D000071231:Carbonic Anhydrase IX / Q000235:genetics* / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003110:Colonic Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D015536:Down-Regulation; D005260:Female; D000072669:Gene Editing; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008819:Mice, Nude; D015398:Signal Transduction; D000074082:Sodium-Hydrogen Exchanger 1 / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D047368:Tumor Burden; D000072258:Tumor Hypoxia; D059016:Tumor Microenvironment
chemical_list: D000951:Antigens, Neoplasm; C490998:SLC9A1 protein, human; D000074082:Sodium-Hydrogen Exchanger 1; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases; C477378:carbonic anhydrase XII
keywords: Carbonic Anhydrase 9 (CA9); Na+/H+-exchanger (NHE1); pH regulation; tumor growth; tumor hypoxia