title: Dentin sialophosphoprotein (DSPP) gene-silencing inhibits key tumorigenic activities in human oral cancer cell line, OSC2.
abstract: BACKGROUND
  We determined recently that dentin sialophosphoprotein (DSPP), a member of the SIBLING (Small integrin-binding ligand N-linked glycoproteins) family of phosphoglycoproteins, is highly upregulated in human oral squamous cell carcinomas (OSCCs) where upregulation is associated with tumor aggressiveness. To investigate the effects of DSPP-silencing on the tumorigenic profiles of the oral cancer cell line, OSC2, short-hairpin RNA (shRNA) interference was employed to silence DSPP in OSC2 cells.
  
  
  RESULTS
  Multiple regions of DSPP transcript were targeted for shRNA interference using hDSP-shRNA lentiviral particles designed to silence DSPP gene expression. Control shRNA plasmid encoding a scrambled sequence incapable of degrading any known cellular mRNA was used for negative control. Following puromycin selection of stable lines of DSSP-silenced OSC2 cells, phenotypic hallmarks of oral carcinogenesis were assayed by western blot and RT-PCR analyses, MTT (cell-viability), colony-formation, modified Boyden-Chamber (migration and invasion), and flow cytometry (cell-cycle and apoptosis) analyses. DSPP-silenced OSC2 cells showed altered cell morphology, reduced viability, decreased colony-formation ability, decreased migration and invasion, G0/G1 cell-cycle arrest, and increased tumor cell sensitivity to cisplatin-induced apoptosis. Furthermore, MMP-2, MMP-3, MMP-9, VEGF, Ki-67, p53, and EGFR were down-regulated. There was a direct correlation between the degree of DSPP-silencing and MMP suppression, as indicated by least squares regression: MMP-2 {(y = 0.850x, p<0.001) (y = 1.156x, p<0.001)}, MMP-3 {(y = 0.994x, p<0.001) (y = 1.324x, p = 0.004)}, and MMP-9 {(y = 1.248x, p = 0.005, y = 0.809, p = 0.013)}.
  
  
  CONCLUSIONS
  DSPP-silencing in OSC2 cell decreased salient hallmarks of oral tumorigenesis and provides the first functional evidence of a potential key role for DSPP in oral cancer biology. The down-regulation of MMP-2, MMP-3, MMP-9, p53 and VEGF in DSPP-silenced OSC2 cells provides a significant functional/molecular framework for deciphering the mechanisms of DSPP activities in oral cancer biology.
authors.affiliation: Department of Oral Biology, School of Dentistry, Medical College of Georgia, Augusta, Georgia, United States of America.
authors.forename: Rajeshree
authors.initials: R
authors.lastname: Joshi
authors.forename: Amany
authors.initials: A
authors.lastname: Tawfik
authors.forename: Nneka
authors.initials: N
authors.lastname: Edeh
authors.forename: Veronica
authors.initials: V
authors.lastname: McCloud
authors.forename: Stephen
authors.initials: S
authors.lastname: Looney
authors.forename: Jill
authors.initials: J
authors.lastname: Lewis
authors.forename: Stephen
authors.initials: S
authors.lastname: Hsu
authors.forename: Kalu U E
authors.initials: KU
authors.lastname: Ogbureke
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002465:Cell Movement; D002945:Cisplatin / Q000494:pharmacology; D016326:Extracellular Matrix Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009062:Mouth Neoplasms / Q000235:genetics* / Q000473:pathology; D009374:Neoplasms, Experimental / Q000235:genetics / Q000473:pathology; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000235:genetics* / Q000378:metabolism; D014183:Transplantation, Heterologous; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D015854:Up-Regulation; D000099311:Dentin Sialophosphoprotein
chemical_list: D000970:Antineoplastic Agents; D002945:Cisplatin; D016326:Extracellular Matrix Proteins; D020778:Matrix Metalloproteinase 2; D019278:Matrix Metalloproteinase 3; D020780:Matrix Metalloproteinase 9; D010750:Phosphoproteins; D012795:Sialoglycoproteins; D016159:Tumor Suppressor Protein p53; D000099311:Dentin Sialophosphoprotein