The tumorigenic role of DSPP and its potential regulation of the unfolded protein response and ER stress in oral cancer cells.

Abstract

Dentin sialophosphoprotein (DSPP) is upregulated in various human cancers, including head and neck squamous cell carcinoma. Cancer cells are commonly found under constant endoplasmic reticulum (ER) stress and exhibit increased levels of misfolded proteins, due to gene mutations and a stressful microenvironment. The present study examined the effects of DSPP silencing on the regulation of ER stress and the unfolded protein response (UPR) in oral cancer cells. A recently established stable DSPP short hairpin (sh)RNA-silenced OSC2 oral cancer cell line was used. The mRNA expression levels of ER stress-associated proteins, including 78kDa glucose-regulated protein (GRP78), sarcoplasmic/endoplasmic reticulum calcium ATPase 2b(SERCA2b), inositol 1,4,5-trisphosphate receptor (IP3r), protein kinase R-like endoplasmic reticulum kinase(PERK), serine/threonine-protein kinase/endoribonuclease IRE1 (IRE1), activating transcription factor6 (ATF6) and matrix metalloproteinase20(MMP20), were assessed by reverse transcription-quantitative polymerase chain reaction. The expression levels of apoptosis-related [B‑cell lymphoma2 (Bcl2), Bcl2-associated Xprotein(Bax) and cytochromec] and cell proliferation-related [proliferating cell nuclear antigen (PCNA)] proteins were analyzed by western blotting. Cell viability, apoptosis and migration were monitored by MTT assay, AnnexinV-fluorescein isothiocyanate flow cytometry and wound-healing assay, respectively. In transiently transfected puromycin‑free OSC2 cells, DSPP silencing markedly downregulated the mRNA expression levels of major ER stress regulators, including GRP78, SERCA2b, PERK, IRE1 and ATF6, as well as MMP20. DSPP silencing also resulted in decreased cell viability and migration, and enhanced apoptosis. Furthermore, PCNA and Bcl2 levels were decreased, whereas Bax and cytochromec protein levels were increased in DSPP-silenced OSC2 cells. Sustained puromycin treatment partially counteracted the effects of DSPP silencing on the mRNA expression levels of ER stress-related proteins and MMP20, and on the migratory capacity of OSC2 cells. However, following puromycin treatment of DSPP-silenced cells, cell viability was further reduced and apoptosis was enhanced. In conclusion, these data provide evidence to suggest that DSPP may be involved in ER stress mechanisms in oral squamous cell carcinoma, since its downregulation in OSC2 cells led to significant alterations in the levels of major ER stress-associated proteins, and subsequent collapse of the UPR system.