| BackgroundNasopharyngeal carcinoma (NPC) is one of the most common forms of clinical malignant cancer, with an incidence of up to 25 per 100,000 individuals per year. It was distributed unevenly in population, and it was most popular in southeast Asian and the southern coastal area of china, such as Guangdong, Guangxi, Fujian and Hongkong. Hence, it was the critical risk factor for human health in china.98% NPC were poorly-differentiated carcinoma, characterized by rapid growth, early distant metastasis, and were sensitive to radiation. With the development of precise radiation technology, the 5-year overall survival rate of patients with NPC has increased to between 67% and 80%. Regrettably, a few patients still relapse or metastasize soon after therapy, while others are diagnosed with distant metastasis, do not qualify for curative treatment and experience poor prognosis. However, the specific development and progression mechanisms in NPC have not yet been completely understood. Therefore, to improve the prognosis of NPC, there is an urgent need to clarify underlying mechanisms of NPC, identify diagnosis/prognosis-related biomarkers, and determine new therapeutic targets for treatment.It is well known that the development and progression of NPC was closely correlated with EB virus, ethnic group, and geographical reason, et.al. Recent studies suggested that some oncogene/suppressor mutation or aberrant expression could involved in malignant phenotype of NPC, and played important role in the development and progression of NPC. Therefore, these functional genes studies in NPC become the focus issue. Ubiquitin-proteasome system exert important functions in many cellular biology process. It was composed by three different enzyme systems. Under normal condition, Ubiquitin activating enzyme El promote the binding of ubiquitin to ubiquitin-conjugating enzyme, while ATP was presented. Subsequently, ubiquitin-conjugating enzyme E2 cooperated with E3 ligase led to mono-ubiquitination or multi-ubiquitination of specific substrate, and resulted in the specific substrate degradation or functional change. The mutation or aberrant expression of these genes encoding these enzyme system proteins were implicated in various malignant cancer.Ubiquitin-conjugating enzyme E2T was a member of Ubiquitin-conjugat ing enzyme E2, and possess a 16-18kDa characteristic conserved Ubiquitin-conjugating doamin-UBC domain. It was first identified in a case of Fanconi anemia, which is characterized by bone marrow failure and predisposition to development of acute myeloid as well as head and neck squamous carcinomas, and is considered to be mainly caused by the dysregulation of a DNA damage-repair pathway, called the Fanconi anemia (FA) pathway (FANCA、 FANCB、 FANCC、 FANCE、 FANCF、 FANCG、 FANCL、 FANCM). UBE2T is a component of this pathway. Recent studies have shown that disruption of UBE2T expression could directly lead to Fanconi anemia as well as an increase in tumor cell sensitivity to crossing-link agents by interfering with the DNA damage-repair response. It suggests that UBE2T play important role in genomic integrity.In addition to the role of UBE2T playing in maintaining the integrity genome, some recent studies showed that UBE2T was high expressed in various cancer tissues than in the adjacent normal tissues, such as lung cancer,prostate cancer and breast cancer, and it was closely correlated with the tumor size, metastasis and poor prognosis. These results suggest that UBE2T might possess the pro-proliferation/invasion/metastasis potency. The further studies showed that UBE2T overexpression promoted the colony formation of NIH3T3 cell lines, and that UBE2T promote breast carcinogenesis via degradation of BRCA1. In addition, Wen et al study demonstrated that UBE2T promoted prostate cancer cells proliferation, EMT, and metastasis through cooperating with vimintin. Considering the vital role of UBE2T playing in genome integrity and various cancer types, we try to explore the correlation of UBE2T with the malignant characteristics of NPC patients, and to find the effects of UBE2T on the proliferation and metastasis, as well as the possible mechanism. All of these will provide theoretical basis for us to take UBE2T as a alternative diagnostic/ prognostic bio-marker and treatment target in NPC.In the present study, the expression of UBE2T in 149 NPC patient specimens were detected through immunohistochemistry (IHC) to analyze the correlation of UBE2T expression and the clinical parameters. Proliferation, invasion, and metastasis assays in vitro and in vivo were performed to define the functions of UBE2T. West blotting and immunofluoresence were used to determine possible mechanisms.Methods:1. Analysis of UBE2T expression in 149 NPC patient samples.We detected the expression of UBE2T in paraffin-embedded samples of 149 patients with NPC by IHC. The expression of UBE2T was score by IRS (immunoreactive score) to elevate the expression of UBE2T in NPC tissues and adjacent normal tissues. According to the IRS, the NPC patients were divide into UBE2T low expression group and UBE2T high expression group. The correlation of UBE2T expression and the clinical parameters were analyzed by pearson correlation analysis. Kaplan-Meier survival analysis to explore the influence of UBE2T expression level on the prognosis of NPC. Univariate and multivariate Cox regression analysis detected the prognostic risk of the malignant characteristics and UBE2T expression. Significant variables in univariate analysis were then included in multivariate regression analysis.2. Explore the effects of UBE2T on proliferation and metastasis of NPC cell in vitro and in vivo.2.1 To explore pro-proliferation effects of UBE2T on NPC cell in vitro and in vivo.We screened the endgenous UBE2T expression in a panel of NPC cells, and found that UBE2T was relative low expression in C666-1 NPC cells and relative high expression in 5-8F、 6-10B、 CNE1 and CNE2 cell lines. We chose C666-1 cell for UBE2T over expression by UBE2T lentivirus and CNE2 for UBE2T silence by siRNA or shUBE2T lentivirus. The pro-proliferation effect of UBE2T in C666-1 cells was detected through MTT, and the proliferation inhibitory effect of UBE2T in CNE2 cells was detected by CCK8. The colony formation assay was using to detected the effect of UBE2T on colony formation ability in C666-1 cells. For detecting the effect of UBE2T on C666-1 cells in vivo, we co-transfected UBE2T and Luciferase lentivirus to C666-1 NPC cells. Subsequently, UBE2T overexpression and control C666-1 cells were injected into the bilaterial flanks of BALB/c nude mice to establish subcutanous xogengraft model. The fluorescence absorption of xogengraft was monitored using the IVIS Lumina Ⅱ system. Moreover, the ki-67 expression of xogengraft was detected by IHC. For detecting the effects of UBE2T on CNE2 cell, UBE2T of CNE2 cells were silenced by shUBE2T lentivirus. UBE2T knockdown CNE2 cells with shRNA lentivirus and the control were inject into bilatiral flanks of BALB/c nude mice to established xogengraft model.2.2 To explore the pro-metastasis effect of UBE2T on NPC cell in vitro and in vivoOn the basis of UBE2T overexpression and knockdown NPC cell lines with UBE2T lentivirus overexpression or siRNA UBE2T silence, we proformed scratch assay to detect the effects of UBE2T overexpression or knockdown on the migration ability of C666-1 and CNE2. Matrix-cocated transwell assay were performed to detect the effects of UBE2T overexpression or knockdown on the invasion ability of C666-1 or CNE2. UBE2T and luciferase lentivirus co-transfected C666-1 were tail vein injected to detecte the effect of UBE2T on the metastasis ability of NPC cells in vivo.3. To explore the pro-proliferation/metastasis mechanisms of UBE2T on NPC cell linesWestern blot were used to detected the effects of UBE2T overexpression and knockdown on proliferation/metastasis-related proteins (Cyclin D1、C-JUN、C-MYC、 MMP2、MMP9) and AKT/GSK3β/β-catenin pathway proteins (p-AKT、p-GSK3β、 β-catenin) in C666-1 or CNE2. Immunofluoreccent technology and nucleoplasm western blot were used to detected the effects of UBE2T overexpression on AKT/GSK3β/β-catenin pathway in C666-1 cells. We detected the blocking effects of MK-2206 2HC1 (a specific inhibitor of AKT) on pro-migration/metastasis and AKT/GSK3β/β-catenin pathway activation effects by UBE2T with transwell and western blot.Rusults:1. UBE2T expression was correlated with malignant characteristics and outcome of NPC patientsTo investigate UBE2T expression in NPC tissues, we evaluated UBE2T levels in paraffin-embedded samples from 149 patients with NPC by IHC. UBE2T was variably expressed in the cytoplasm of tumor cells in 140 out of the 149 samples, with higher expression in the peripheral region than in the central region of the typical cancer nest. However, only weak expression was noted in 10 out of the 90 samples of adjacent normal tissue, especially in the basilar membrane cells of normal nasopharyngeal mucosa. Chi-square analysis showed that the UBE2T positive-expression ratio in tumor tissue was higher than that in adjacent normal tissue (P<0.001). Next, we analyzed the association between UBE2T expression in tumor tissue and clinical pathological parameters of patients with NPC. UBE2T expression was positively correlated with the T classification (P=0.006), M classification (P=0.038), and skull base invasion (P=0.022); however no correlation was observed with gender, age, clinical classification, and recurrence. Meanwhile, we found that patients whose tumor tissue showed high UBE2T expression had shorter overall survival time than those with low UBE2T expression, using Kaplan-Meier analysis and log-rank test (P=0.006). More importantly, univariate and multivariate Cox regression analysis indicated that N/M classification, skull base invasion, and UBE2T expression were each, recognized as independent prognostic factors in patients with NPC.2. UBE2T promoted the proliferation and metastasis of NPC cells in vitro and in vivo2.1 UBE2T promoted NPC cell proliferation in vitro and in vivoTo investigate the biological function of UBE2T in NPC, background expression of UBE2T was determined in a panel of NPC cells by western blot. UBE2T was highly expressed in 5-8F,6-10B, CNE1, and CNE2 cell lines, but showed relatively low expression in C666-1 cells. Subsequently, UBE2T-overexpressing C666-1 cells were established by transfecting UBE2T lentivirus (UBE2T), while UBE2T-knockdown CNE2 cells were established by disrupting endogenous UBE2T with small interfering RNA (siUBE2T). We found that UBE2T-overexpressing C666-1 cells showed a significantly higher proliferation rate than empty vector-transfected control (NC) using the MTT assay (P<0.001). Conversely, UBE2T knockdown inhibited the proliferation of CNE2 cells compared to scrambled UBE2T (Scramble) using the CCK8 assay (P<0.001). In addition, UBE2T promoted colony formation of C666-1 cells compared to the control (P<0.001). These data suggest that UBE2T promoted NPC cell proliferation in vitro. To verify this pro-proliferation activation of UBE2T in vivo, we subcutaneously inoculated UBE2T-and luciferase-co-expressed C666-1 cells into BALB/c nude mice. Changes in the luminescence absorption value of xenografts indicated that xenografts overexpressing UBE2T showed faster growth than the control (.P<0.001). Moreover, the tumor xenografts were larger and heavier than the control (P=0.024). The number of cells showing Ki-67 positivity (a proliferation-related marker) in xenografts overexpressing UBE2T was much higher than in the control (P<0.001). Nevertheless, silencing UBE2T using shRNA lentivirus (shUBE2T) inhibited the growth of CNE2 subcutaneous xenografts compared to the control (P<0.001). Overall, these findings indicate that UBE2T could facilitate NPC cell proliferation in vitro and in vivo.2.2 UBE2T enhanced invasive and metastatic capacities of NPC cells in vitro and in vivoWe found that UBE2T high expression correlated with skull base invasion and metastasis in patients with NPC. Next, we investigated whether UBE2T promoted NPC cell invasion and metastasis. Using scratch and matrix-coated transwell assays, we found that overexpression of UBE2T significantly promoted C666-1 cell migration and invasion, whereas UBE2T knockdown inhibited CNE2 migration and invasion in vitro. Further in vivo studies showed that UBE2T improved C666-1 cell metastasis in BALB/c nude mice, as indicated by the total luminescence absorption in tumor cells (total luminescence absorption=the sum of all absorption values (P =0.034). Verified by hematoxylin-eosin staining, metastasis organs in the UBE2T overexpressed group were higher than that in the control group. These results demonstrate that invasive and metastatic capacities of NPC cells were enhanced by UBE2T in vitro and in vivo.3. UBE2T promotes NPC cell proliferation and metastasis by activating AKT/GSK3β/β-catenin pathwayWe explored whether UBE2T promoted NPC cells proliferation and metastasis by mediating the β-catenin pathway. We found that UBE2T overexpression in C666-1 elevated protein levels of β-catenin and promoted β-catenin downstream proliferation/metastasis-related target proteins CyclinDl, C-MYC, C-JUN, MMP2, and MMP9 expression, while increasing upstream phosphorylated AKT and GSK3β levels, as compared to the control, by western blot analysis. On the other hand, UBE2T knockdown resulted in decrease of AKT/GSK3β/β-catenin pathway-related protein and β-catenin-related target protein expression. Additionally, immunofluorescence staining results demonstrated that UBE2T promoted the accumulation of β-catenin in the nucleus of C666-1 cells; this was verified by separate western blot analysis of nuclear and cytoplasmic proteins. MK-2206 2HC1 (an specific inhibitor of AKT) blocked the pro-migration/invasion effects (all P< 0.001) and the activating of AKT/GSK3β/β-catenin pathway resulted from UBE2T overexpression, as confirmed via transwell analysis and western blot. Collectively, these results suggest that UBE2T might promote NPC cell proliferation and metastasis via modulating the AKT/GSK3β/β-catenin pathway. To validate this conclusion, we performed IHC analysis on serial sections of 20 additional NPC samples for UBE2T and p-GSK3β expression. The result showed that UBE2T and p-GSK3β were co-expressed in these samples, and the expressions are correlated.Conclusion:1. UBE2T was higher expression in the cancer tissues than adjacent normal tissues; the higher expression of UBE2T was positively correlated with the malignant and poor prognosis in patients with NPC, and was the independent prognostic risk factor. Hence, UBE2T could be take as a alternative diagnostic/prognostic bio-marker.2. UBE2T promotes the proliferation, invasion and metastasis of NPC cell lines in vivo and in vitro. It suggested that UBE2T can serve as a novel molecular target of NPC.3. UBE2T promotes proliferation, invasion and metastasis of NPC via activating the Akt/GSK-3β/β-catenin pathway. It further suggested that UBE2T can serve as a novel molecular target of NPC for in-depth study. |
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