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 78 kDa 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 factor 6 (ATF6) and matrix metalloproteinase 20 (MMP20), were assessed by reverse transcription-quantitative polymerase chain reaction. The expression levels of apoptosis-related [B‑cell lymphoma 2 (Bcl2), Bcl2-associated X protein (Bax) and cytochrome c] 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, Annexin V-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 cytochrome c 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.

Interferon γ suppresses dentin sialophosphoprotein in oral squamous cell carcinoma cells resulting in antitumor effects, via modulation of the endoplasmic reticulum response

“…Our previous report revealed that the strong binding of DSP to MMP20, and its interaction with the promoter proximal element of MMP20, may account for increased migration, invasion and metastasis in OSCC ( 32 ). Recently, we reported that DSPP silencing results in significantly reduced MMP20 mRNA expression and in reduced migration of OSCC cells ( 34 ). Other investigators have reported that IFNγ and IFNβ suppress MMP9 expression through a signal transducer and activator of transcription (STAT)1α pathway in primary astrocytes and human fibrosarcoma cells ( 36 ), and that IFNγ treatment reduces migration of A172 human glioblastoma cells ( 37 ).…”

“…Western blot analysis was performed as previously described ( 34 ). Briefly, cells were lysed and sonicated (20 kHz, 2×10 sec) in radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.).…”

“…In addition, DSPP silencing increases cisplatin sensitivity and enhances apoptosis of OSCC cells, whereas subcutaneous injection of OSCC xenografted Balb/c nude mice with DSPP-silenced OSCC cells results in attenuated tumor growth ( 33 ). Our recent report proposed a tumorigenic role for DSPP in OSCC cells, and presented a relationship between DSPP and the ER chaperone GRP78 ( 34 ). Furthermore, our report suggested a DSPP-associated modulatory effect on ER stress, Ca homeostasis and UPR proteins, including sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA2b), IRE1, PERK and ATF6 ( 34 ).…”

“…Our recent report proposed a tumorigenic role for DSPP in OSCC cells, and presented a relationship between DSPP and the ER chaperone GRP78 ( 34 ). Furthermore, our report suggested a DSPP-associated modulatory effect on ER stress, Ca homeostasis and UPR proteins, including sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA2b), IRE1, PERK and ATF6 ( 34 ).…”

Use of signals of positive and negative selection to distinguish cancer genes and passenger genes

“…There is evidence in the scientific literature that the protein may have a tumorigenic role in oral cancer (Chaplet et al, 2006;Johi et al, 2010;Saxena et al, 2015;Gkouveris et al, 2018;Nikitakis et al, 2018). Nevertheless, the high silent to missense rate is not a reflection of the importance of DSPP for carcinogenesis.…”