title: Prognostic relevance of carbonic anhydrase IX expression is distinct in various subtypes of breast cancer and its silencing suppresses self-renewal capacity of breast cancer cells.
abstract: OBJECTIVE
  Carbonic anhydrase IX (CAIX) is a hypoxia-inducible enzyme with extracellular catalytic domain that is overexpressed in a variety of cancers including breast cancer and plays a crucial role in maintaining favourable intracellular pH and reducing extracellular pH. The purpose of the current study was to elucidate the prognostic significance of CAIX in the intrinsic subtypes of breast cancer and to characterise CAIX as a drug target in breast cancer.
  
  
  METHODS
  The prognostic significance of CAIX mRNA expression was interrogated in a cohort of 3,455 breast tumours by using an online tool, Kaplan-Meier plotter. The functional effects of stable CAIX depletion by shRNA in three breast cancer cell lines—MDA-MB-231, MCF7 and SKBR-3, representing basal-like, luminal A and HER2+ subtypes, respectively—were studied by proliferation, invasion, clonal spheroid formation and chemosensitivity assays under normoxia and hypoxia. Finally, the effect of pharmacological CA inhibition alone or in the combination with doxorubicin on self-renewal was assessed by spheroid-forming assay.
  
  
  RESULTS
  High CAIX mRNA expression was significantly associated with poor survival in patients with basal-like, luminal B and triple-negative breast cancer, but not luminal A and HER+ subtypes. Silencing of CAIX expression had no significant effect on the cell proliferation or viability upon treatment with doxorubicin in any of the cell lines studied, while it inhibited spheroid formation in hypoxic conditions. Furthermore, pharmacological inhibition of CAs using acetazolamide had a synergistic effect with doxorubicin on decreasing the spheroid-forming efficiency in MDA-MB-231 cells.
  
  
  CONCLUSIONS
  Inhibition of CAIX reduces the self-renewal capacity of breast cancer cells, and the combination of doxorubicin and CAIX inhibition is an attractive therapeutic strategy in basal-like and triple-negative breast cancer, which warrants further investigations.
authors.affiliation: Latvian Biomedical Research and Study Centre, Ratsupites Str 1, Riga, 1067, Latvia.
authors.forename: Lāsma
authors.initials: L
authors.lastname: Ivanova
authors.forename: Elīna
authors.initials: E
authors.lastname: Zandberga
authors.forename: Karīna
authors.initials: K
authors.lastname: Siliņa
authors.forename: Zane
authors.initials: Z
authors.lastname: Kalniņa
authors.forename: Artūrs
authors.initials: A
authors.lastname: Ābols
authors.forename: Edgars
authors.initials: E
authors.lastname: Endzeliņš
authors.forename: Ineta
authors.initials: I
authors.lastname: Vendina
authors.forename: Nadezhda
authors.initials: N
authors.lastname: Romanchikova
authors.forename: Alinta
authors.initials: A
authors.lastname: Hegmane
authors.forename: Pēteris
authors.initials: P
authors.lastname: Trapencieris
authors.forename: Jānis
authors.initials: J
authors.lastname: Eglītis
authors.forename: Aija
authors.initials: A
authors.lastname: Linē
mesh_terms: D000951:Antigens, Neoplasm / Q000032:analysis* / Q000235:genetics*; D014408:Biomarkers, Tumor / Q000032:analysis*; D001943:Breast Neoplasms / Q000235:genetics*; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000032:analysis* / Q000235:genetics*; D045744:Cell Line, Tumor; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D020868:Gene Silencing; D006801:Humans; D053208:Kaplan-Meier Estimate; D011237:Predictive Value of Tests; D011379:Prognosis; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics; D018874:Spheroids, Cellular / Q000187:drug effects
chemical_list: D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human; C089226:CA9 protein, human