title: Chondroitin sulfates play a major role in breast cancer metastasis: a role for CSPG4 and CHST11 gene expression in forming surface P-selectin ligands in aggressive breast cancer cells.
abstract: BACKGROUND
  We have previously demonstrated that chondroitin sulfate glycosaminoglycans (CS-GAGs) on breast cancer cells function as P-selectin ligands. This study was performed to identify the carrier proteoglycan (PG) and the sulfotransferase gene involved in synthesis of the surface P-selectin-reactive CS-GAGs in human breast cancer cells with high metastatic capacity, as well as to determine a direct role for CS-GAGs in metastatic spread.
  
  
  METHODS
  Quantitative real-time PCR (qRT-PCR) and flow cytometry assays were used to detect the expression of genes involved in the sulfation and presentation of chondroitin in several human breast cancer cell lines. Transient transfection of the human breast cancer cell line MDA-MB-231 with the siRNAs for carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) and chondroitin sulfate proteoglycan 4 (CSPG4 ) was used to investigate the involvement of these genes in expression of surface P-selectin ligands. The expression of CSPG4 and CHST11 in 15 primary invasive breast cancer clinical specimens was assessed by qRT-PCR. The role of CS-GAGs in metastasis was tested using the 4T1 murine mammary cell line (10 mice per group).
  
  
  RESULTS
  The CHST11 gene was highly expressed in aggressive breast cancer cells but significantly less so in less aggressive breast cancer cell lines. A positive correlation was observed between the expression levels of CHST11 and P-selectin binding to cells (P < 0.0001). Blocking the expression of CHST11 with siRNA inhibited CS-A expression and P-selectin binding to MDA-MB-231 cells. The carrier proteoglycan CSPG4 was highly expressed on the aggressive breast cancer cell lines and contributed to the P-selectin binding and CS-A expression. In addition, CSPG4 and CHST11 were over-expressed in tumor-containing clinical tissue specimens compared with normal tissues. Enzymatic removal of tumor-cell surface CS-GAGs significantly inhibited lung colonization of the 4T1 murine mammary cell line (P = 0.0002).
  
  
  CONCLUSIONS
  Cell surface P-selectin binding depends on CHST11 gene expression. CSPG4 serves as a P-selectin ligand through its CS chain and participates in P-selectin binding to the highly metastatic breast cancer cells. Removal of CS-GAGs greatly reduces metastatic lung colonization by 4T1 cells. The data strongly indicate that CS-GAGs and their biosynthetic pathways are promising targets for the development of anti-metastatic therapies.
authors.affiliation: Department of Biochemistry and Molecular Biology, the University of Arkansas for Medical Sciences, Little Rock, 72205, USA.
authors.forename: Craig A
authors.initials: CA
authors.lastname: Cooney
authors.forename: Fariba
authors.initials: F
authors.lastname: Jousheghany
authors.forename: Aiwei
authors.initials: A
authors.lastname: Yao-Borengasser
authors.forename: Bounleut
authors.initials: B
authors.lastname: Phanavanh
authors.forename: Tina
authors.initials: T
authors.lastname: Gomes
authors.forename: Ann Marie
authors.initials: AM
authors.lastname: Kieber-Emmons
authors.forename: Eric R
authors.initials: ER
authors.lastname: Siegel
authors.forename: Larry J
authors.initials: LJ
authors.lastname: Suva
authors.forename: Soldano
authors.initials: S
authors.lastname: Ferrone
authors.forename: Thomas
authors.initials: T
authors.lastname: Kieber-Emmons
authors.forename: Behjatolah
authors.initials: B
authors.lastname: Monzavi-Karbassi
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D011508:Chondroitin Sulfate Proteoglycans / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002809:Chondroitin Sulfates / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009362:Neoplasm Metastasis; D019007:P-Selectin / Q000235:genetics / Q000378:metabolism*; D011509:Proteoglycans / Q000032:analysis; D034622:RNA Interference; D034741:RNA, Small Interfering; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*; D000099216:Chondroitin Sulfate Proteoglycan 4
chemical_list: D011508:Chondroitin Sulfate Proteoglycans; D002809:Chondroitin Sulfates; D008024:Ligands; D008565:Membrane Proteins; D019007:P-Selectin; D011509:Proteoglycans; D034741:RNA, Small Interfering; D015238:Sulfotransferases; C101390:CSPG4 protein, human; D000099216:Chondroitin Sulfate Proteoglycan 4; C411659:CHST11 protein, human