title: Cathepsin K induces platelet dysfunction and affects cell signaling in breast cancer - molecularly distinct behavior of cathepsin K in breast cancer.
abstract: BACKGROUND
  Breast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes to invasiveness. Although proteolytically activated receptors (PARs) are activated by proteases, the direct interaction of cysteine cathepsins with PARs is poorly understood. In human platelets, PAR-1 and -4 are highly expressed, but PAR-3 shows low expression and unclear functions.
  
  
  METHODS
  Platelet aggregation was monitored by measuring changes in turbidity. Platelets were immunoblotted with anti-phospho and total p38, Src-Tyr-416, FAK-Tyr-397, and TGFβ monoclonal antibody. Activation was measured in a flow cytometer and calcium mobilization in a confocal microscope. Mammary epithelial cells were prepared from the primary breast cancer samples of 15 women with Luminal-B subtype to produce primary cells.
  
  
  RESULTS
  We demonstrate that platelets are aggregated by cathepsin K in a dose-dependent manner, but not by other cysteine cathepsins. PARs-3 and -4 were confirmed as the cathepsin K target by immunodetection and specific antagonists using a fibroblast cell line derived from PARs deficient mice. Moreover, through co-culture experiments, we show that platelets activated by cathepsin K mediated the up-regulation of SHH, PTHrP, OPN, and TGFβ in epithelial-mesenchymal-like cells from patients with Luminal B breast cancer.
  
  
  CONCLUSIONS
  Cathepsin K induces platelet dysfunction and affects signaling in breast cancer cells.
authors.affiliation: Departments of Gynecology of Universidade Federal de São Paulo, São Paulo, SP, 04024-002, Brazil. sheilasa@gmail.com.|Charitable Association of Blood Collection - COLSAN, São Paulo, SP, 04080-006, Brazil. sheilasa@gmail.com.|Department of Gynecology, Cellular Gynecology Laboratory, Universidade Federal de São Paulo, Rua Napoleão de Barros, 608, CEP 04024-002, São Paulo, Brazil. sheilasa@gmail.com.
authors.forename: Sheila Siqueira
authors.initials: SS
authors.lastname: Andrade
authors.affiliation: Biophysics of Universidade Federal de São Paulo, São Paulo, SP, 04024-002, Brazil. iurig@yahoo.com.
authors.forename: Iuri Estrada
authors.initials: IE
authors.lastname: Gouvea
authors.affiliation: Biochemistry of Universidade Federal de São Paulo, São Paulo, SP, 04024-002, Brazil. mccscris@ig.com.br.
authors.forename: Mariana Cristina C
authors.initials: MC
authors.lastname: Silva
authors.affiliation: Biochemistry of Universidade Federal de São Paulo, São Paulo, SP, 04024-002, Brazil. elocastro31@gmail.com.
authors.forename: Eloísa Dognani
authors.initials: ED
authors.lastname: Castro
authors.affiliation: Biochemistry of Universidade Federal de São Paulo, São Paulo, SP, 04024-002, Brazil. claulondrina@yahoo.com.br.
authors.forename: Cláudia A A
authors.initials: CA
authors.lastname: de Paula
authors.affiliation: Biophysics of Universidade Federal de São Paulo, São Paulo, SP, 04024-002, Brazil. deboraok@yahoo.com.br.
authors.forename: Debora
authors.initials: D
authors.lastname: Okamoto
authors.affiliation: Biophysics of Universidade Federal de São Paulo, São Paulo, SP, 04024-002, Brazil. lilian_cgo@yahoo.com.br.
authors.forename: Lilian
authors.initials: L
authors.lastname: Oliveira
authors.affiliation: Biochemistry of Universidade Federal de São Paulo, São Paulo, SP, 04024-002, Brazil. giovaniperes@gmail.com.
authors.forename: Giovani Bravin
authors.initials: GB
authors.lastname: Peres
authors.affiliation: Biochemistry of Universidade Federal de São Paulo, São Paulo, SP, 04024-002, Brazil. tatiana.ottaiano@gmail.com.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Ottaiano
authors.affiliation: Departments of Gynecology of Universidade Federal de São Paulo, São Paulo, SP, 04024-002, Brazil. gilfacina@hotmail.com.
authors.forename: Gil
authors.initials: G
authors.lastname: Facina
authors.affiliation: Departments of Gynecology of Universidade Federal de São Paulo, São Paulo, SP, 04024-002, Brazil. nazario.gineco@epm.br.
authors.forename: Afonso Celso Pinto
authors.initials: AC
authors.lastname: Nazário
authors.affiliation: Department of Pathology, AC Camargo Hospital Biobank, A C Camargo Cancer Center - Antonio Prudente Foundation, São Paulo, SP, 01509-010, Brazil. ahcampos@accamargo.org.br.
authors.forename: Antonio Hugo J F M
authors.initials: AH
authors.lastname: Campos
authors.affiliation: Biochemistry of Universidade Federal de São Paulo, São Paulo, SP, 04024-002, Brazil. paredes.gamero@gmail.com.
authors.forename: Edgar Julian
authors.initials: EJ
authors.lastname: Paredes-Gamero
authors.affiliation: Biophysics of Universidade Federal de São Paulo, São Paulo, SP, 04024-002, Brazil. ma.juliano@unifesp.br.
authors.forename: Maria
authors.initials: M
authors.lastname: Juliano
authors.affiliation: Departments of Gynecology of Universidade Federal de São Paulo, São Paulo, SP, 04024-002, Brazil. ismael.dale@gmail.com.
authors.forename: Ismael D C G
authors.initials: ID
authors.lastname: da Silva
authors.affiliation: Biochemistry of Universidade Federal de São Paulo, São Paulo, SP, 04024-002, Brazil. olivamlv@gmail.com.
authors.forename: Maria Luiza V
authors.initials: ML
authors.lastname: Oliva
authors.affiliation: Departments of Gynecology of Universidade Federal de São Paulo, São Paulo, SP, 04024-002, Brazil. mjbcg@terra.com.br.|Charitable Association of Blood Collection - COLSAN, São Paulo, SP, 04080-006, Brazil. mjbcg@terra.com.br.
authors.forename: Manoel J B C
authors.initials: MJ
authors.lastname: Girão
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001943:Breast Neoplasms / Q000097:blood / Q000378:metabolism* / Q000473:pathology; D002118:Calcium / Q000378:metabolism; D056657:Cathepsin K / Q000378:metabolism* / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D005260:Female; D053823:Hedgehog Proteins / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008024:Ligands; D008565:Membrane Proteins / Q000037:antagonists & inhibitors; D051379:Mice; D010766:Phosphorylation; D015539:Platelet Activation / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D059748:Proteolysis; D018179:Receptors, Thrombin / Q000037:antagonists & inhibitors; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D053823:Hedgehog Proteins; D008024:Ligands; D008565:Membrane Proteins; C509689:PARD3 protein, human; D018179:Receptors, Thrombin; D019061:src-Family Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D013917:Thrombin; D056657:Cathepsin K; C112908:protease-activated receptor 4; D002118:Calcium