title: The basal epithelial marker P-cadherin associates with breast cancer cell populations harboring a glycolytic and acid-resistant phenotype.
abstract: BACKGROUND
  Cancer stem cells are hypoxia-resistant and present a preponderant glycolytic metabolism. These characteristics are also found in basal-like breast carcinomas (BLBC), which show increased expression of cancer stem cell markers.Recently, we demonstrated that P-cadherin, a biomarker of BLBC and a poor prognostic factor in this disease, mediates stem-like properties and resistance to radiation therapy. Thus, the aim of the present study was to evaluate if P-cadherin expression was associated to breast cancer cell populations with an adapted phenotype to hypoxia.
  
  
  METHODS
  Immunohistochemistry was performed to address the expression of P-cadherin, hypoxic, glycolytic and acid-resistance biomarkers in primary human breast carcinomas. In vitro studies were performed using basal-like breast cancer cell lines. qRT-PCR, FACS analysis, western blotting and confocal microscopy were used to assess the expression of P-cadherin after HIF-1α stabilization, achieved by CoCl2 treatment. siRNA-mediated knockdown was used to silence the expression of several targets and qRT-PCR was employed to evaluate the effects of P-cadherin on HIF-1α signaling. P-cadherin high and low breast cancer cell populations were sorted by FACS and levels of GLUT1 and CAIX were assessed by FACS and western blotting. Mammosphere forming efficiency was used to determine the stem cell activity after specific siRNA-mediated knockdown, further confirmed by western blotting.
  
  
  RESULTS
  We demonstrated that P-cadherin overexpression was significantly associated with the expression of HIF-1α, GLUT1, CAIX, MCT1 and CD147 in human breast carcinomas. In vitro, we showed that HIF-1α stabilization was accompanied by increased membrane expression of P-cadherin and that P-cadherin silencing led to a decrease of the mRNA levels of GLUT1 and CAIX. We also found that the cell fractions harboring high levels of P-cadherin were the same exhibiting more GLUT1 and CAIX expression. Finally, we showed that P-cadherin silencing significantly decreases the mammosphere forming efficiency in the same range as the silencing of HIF-1α, CAIX or GLUT1, validating that all these markers are being expressed by the same breast cancer stem cell population.
  
  
  CONCLUSIONS
  Our results establish a link between aberrant P-cadherin expression and hypoxic, glycolytic and acid-resistant breast cancer cells, suggesting a possible role for this marker in cancer cell metabolism.
authors.forename: Bárbara
authors.initials: B
authors.lastname: Sousa
authors.forename: Ana Sofia
authors.initials: AS
authors.lastname: Ribeiro
authors.forename: Ana Rita
authors.initials: AR
authors.lastname: Nobre
authors.forename: Nair
authors.initials: N
authors.lastname: Lopes
authors.forename: Diana
authors.initials: D
authors.lastname: Martins
authors.forename: Céline
authors.initials: C
authors.lastname: Pinheiro
authors.forename: André Filipe
authors.initials: AF
authors.lastname: Vieira
authors.forename: André
authors.initials: A
authors.lastname: Albergaria
authors.forename: René
authors.initials: R
authors.lastname: Gerhard
authors.forename: Fernando
authors.initials: F
authors.lastname: Schmitt
authors.forename: Fátima
authors.initials: F
authors.lastname: Baltazar
authors.affiliation: IPATIMUP- Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr Roberto Frias s/n, Porto 4200-465, Portugal. jparedes@ipatimup.pt.
authors.forename: Joana
authors.initials: J
authors.lastname: Paredes
mesh_terms: D000137:Acid-Base Imbalance / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism*; D015820:Cadherins / Q000378:metabolism*; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism; D018270:Carcinoma, Ductal, Breast / Q000378:metabolism*; D045744:Cell Line, Tumor; D005260:Female; D015870:Gene Expression; D051272:Glucose Transporter Type 1 / Q000378:metabolism; D006019:Glycolysis; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D010641:Phenotype; D018874:Spheroids, Cellular / Q000378:metabolism; D046888:Tissue Array Analysis
chemical_list: D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; C512197:CDH3 protein, human; D015820:Cadherins; D051272:Glucose Transporter Type 1; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C495677:SLC2A1 protein, human; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases