TRB3Due To Endoplasmic Reticulum Stress Induced Apoptosis By Inhibiting AKT Kinase Phosphorylation In Human Tongue Squamous Cell Carcinoma Cells

Objective:Oral tongue squamous cell carcinoma (OTSCC) is the most common kind of oral cancer and has increasing trend in whole world, especially among young adults aged from20to44years. Even with combined treatment of surgery, chemotherapy and radiation, the five-year survival rate of OTSCC is only about60%. Therefore, exploring a new biomarker for OTSCC prognosis and treatment is necessary. TRB3, a mammalian homolog of Drosophila tribbles, has been found overexpressed in kinds of tumor and correlated with their prognosis. Endoplasmic reticulum stress (ERS) is an important form of cells'response to environment stress and becomes common in solid tumor. TRB3is not only overexpressed in tumor, but also plays an important role in ERS. Under a severe or prolonged stress up-regulates TRB3and induces cell apoptosis. TRB protein family has no ATP combining site, and therefore requires a connection with other kinase binding areas to take effects. AKT (a serine/threonine protein kinase) signaling pathway is closely correlated with carcinogenesis and the development of head and neck squamous cell carcinoma Moreover, TRB3inhibits AKT activation through combining with its threonine or serine activated site, resulting in activating the downstream signal to induce cell apoptosis. In this study, first, retrospective analysis of treatment and prognosis of OTSCC was conducted. Second, we detected TRB3and AKT expression level in OTSCC tissues and analyzed its relationship with OTSCC prognosis. Finally, we studied relationship between TRB3expression and AKT phosphorylation in OTSCC cells, discussing molecular mechanisms of apoptosis induced by TRB3.Methods:Part I:254OTSCC patients firstly diagnosed and treatment between January2000and December2005in head and neck surgery department of the cancer center at Sun Yat-sen University in Guangzhou, China were collected. All statistical analyses were carried out using SPSS 16.0statistical software package. Kaplan-Meier method was used to calculate overall cumulative survival time (from treatment time to death or last follow-up time),disease-free survival time (from treatment time to recurrence or metastasis or death or last follow-up time). Survival time was compared by log-rank test. The χ2test for proportion was used to analyze the clinical and pathological characteristics. The significance of variables for survival was analyzed by the Cox proportional hazards model in multivariate analysis. P<0.05was considered statistically significant.Part II:For reverse transcription polymerase chain reaction to detect expression of TRB3mRNA and Western blot analysis to detect expression of TRB3protein and phospho-AKT (p-AKT) protein, we collected18paired OTSCC and adjacent noncancerous tongue mucosa tissues (at a distance greater than2cm from tumor) from patients who underwent tongue surgery between May and July2010in head and neck surgery department of the cancer center at Sun Yat-sen University in Guangzhou, China. In addition,128paraffin-embedded samples of OTSCC and30specimens of adjacent noncancerous tongue mucosa tissues were collected between January2000and December2002for immunohistochemical testing expression of TRB3protein and p-AKT protein. The relationship between TRB3and p-AKT expression level and OTSCC prognosis was analyzed.Part III:In OTSCC cells Tca8113and CAL-27, ERS was induced by thapsigargin or tunicmycin. Real time polymerase chain reaction to detect expression of TRB3mRNA and Western blot analysis to detect expression of TRB3protein and phospho-AKT (p-AKT) protein to confirm TRB3was induced by ERS. TRB3expression was up-regulated by TRB3adenovirus plasmid (Ad-TRB3) transfection and down-regulated by short hairpin RNA (shRNA) inhibition. Then real time polymerase chain reaction to detect expression of TRB3mRNA and Western blot analysis to detect expression of TRB3protein and phospho-AKT (p-AKT) protein to validate correlation of TRB3and AKT phosphorylation.Results:Part I:In254OTSCC cases overall survival rate at3and5years was71.4%,66.1%and disease-free survival rate was67.3%,53.3%. Overall survival rate at5years was89.6%in68clinical I stage OTSCC cases and68.2%in103clinical Ⅱ stage OTSCC cases, which had significant difference(P=0.002). Overall survival rate at5years was51.4%in66clinical III stage OTSCC cases, but in17clinicalⅣ stage OTSCC cases overall survival rate at3years was5.9%, which had significant difference (P<0.001). In III and IV stage cases,31cases treated with surgery only, overall survival rate at5years was57.7%.26cases treated with surgery and chemotheraphy (or radiatheraphy), overall survival rate at5years was41.4%.9cases treated with chemotheraphy and radiatheraphy, overall survival rate at3years was6.2%.The survival rates of3different treatment had significant difference(P<0.001). In addition, Cox regression analysis indicated that T stage, N stage, histological grade and recurrence were independent predictors of OTSCC survival.Part II:In18paired tissues, expression of TRB3mRNA in15(83.3%) OTSCC tissues were significantly higher than adjacent noncancerous mucosa. And expression of TRB3protein and p-AKT protein were both significantly higher in OTSCC tissue than adjacent noncancerous mucosa in13/18(72.2%) Western blot assays. Also immunohistochemical assays showed high expression of TRB3protein was in63/128(49.2%) and high expression of p-AKT protein in88/128(68.8%) paraffin-embedded OTSCC samples, expression of TRB3and p-AKT were significantly higher in OTSCC tissue than adjacent noncancerous tissue (both P≤0.001), both of which were significantly correlated with tumor pathological T stage, lymph grade and tumor recurrence. The expression level of TRB3protein showed a significant negative correlation with the expression level of p-AKT (r=-0.27, P<0.01)Part III:In OTSCC cell lines Tca8113and CAL-27, both of the mRNA and protein expression of TRB3were induced by thapsigargin (5mol/1) or tunicmycin (5g/ml) for24hours.. The expression of p-AKT was decreased under Ad-TRB3transfection-induced TRB3overexpression in Tca8113cells. Furthermore, the p-AKT expression was increased by suppressing TRB3expression through shRNA inhibition.Conclusions:1,The most important treatment of OTSCC was surgery, including high-risk cases added with chemotheraphy and radiotheraphy. T stage, N stage, histological grade and recurrence were independent predictors of OTSCC survival.2,TRB3and p-AKT was overexpressed in OTSCC and was linked to tumor prognosis. Expression of p-AKT was correlated negatively expression of TRB3.3,When ERS was induced TRB3protein expression was up-regulated and inhibited AKT phosphorylation of OTSCC. Our finding provided a new treatment target for OTSCC.