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.

METHODOLOGY/PRINCIPAL FINDINGS:

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/SIGNIFICANCE:

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: Rajeshree Joshi, Amany Tawfik, Nneka Edeh, Veronica McCloud, Stephen Looney, Jill Lewis, Stephen Hsu, Kalu U E Ogbureke
• Journal: PloS one (PLoS One) Vol. 5 Issue 11 Pg. e13974 (Nov 12 2010) ISSN: 1932-6203 [Electronic] United States
• PMID: 21103065 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Antineoplastic Agents
• Extracellular Matrix Proteins
• Phosphoproteins
• Sialoglycoproteins
• Tumor Suppressor Protein p53
• dentin sialophosphoprotein
• Matrix Metalloproteinase 3
• Matrix Metalloproteinase 2
• Matrix Metalloproteinase 9
• Cisplatin

Topics

• Animals
• Antineoplastic Agents (pharmacology)
• Apoptosis (drug effects)
• Blotting, Western
• Cell Line
• Cell Line, Tumor
• Cell Movement
• Cisplatin (pharmacology)
• Extracellular Matrix Proteins (genetics, metabolism)
• Female
• Humans
• Keratinocytes (cytology, metabolism)
• Matrix Metalloproteinase 2 (metabolism)
• Matrix Metalloproteinase 3 (metabolism)
• Matrix Metalloproteinase 9 (metabolism)
• Mice
• Mice, Inbred BALB C
• Mice, Nude
• Mouth Neoplasms (genetics, pathology)
• Neoplasms, Experimental (genetics, pathology)
• Phosphoproteins (genetics, metabolism)
• RNA Interference
• Reverse Transcriptase Polymerase Chain Reaction
• Sialoglycoproteins (genetics, metabolism)
• Transplantation, Heterologous
• Tumor Suppressor Protein p53 (metabolism)
• Up-Regulation