Molecular Mechanism And Clinical Study On Distant Metastasis Of Nasopharyngeal Carcinoma Treated With Accurate Radiotherapy

Purpose: This study aimed to evaluate the survival outcomes and the failure patterns of patients with nasopharyngeal carcinoma(NPC) by intensity-modulated radiotherapy(IMRT), and explore the molecular mechanisms and clinical risk factors of distant-metastasis(DM). Our preliminary work found that MAZ presented high expression in NPC distant metastasis tissue by high-throughput genome-wide microarray. However, whether MAZ expression were connected with NPC distant-metastasis after IMRT radiotherapy remains unclear. MAZ expression were assayed by immunohistochemistry in metastatic nasopharyngeal carcinoma tissue samples,and we explore the correlation between MAZ expression and clinical characteristic in metastatic nasopharyngeal carcinoma. Real-time PCR and Western Blot assay MAZ expression in high and low metastatic cell lines, MAZ expression promoted nasopharyngeal carcinoma cell metastasis and invasion,further revealed molecular mechanisms of MAZ in NPC metastasis, which could provide new targets and strategies for prevention and treatment of NPC.Method:1. All patients were native and resided in northwest China, consisted of 233 consecutive untreated patients who had histologically confirmed NPC before radiotherapy between January 2006 and October 2011. MRI/CT images was performed for all patients for accurate delineation of tumor volumes and critical structures. Univariate and Cox regression models were used to analysis risk factors,Survival analyses were computed using the Kaplan-Meier method. 2. Collecting 80 tissue specimens of patients with nasopharyngeal carcinoma, to analysis correlation between MAZ expression and clinical data or prognosis. immunohistochemistry assay MAZ expression. Real-time PCR and Western Blot assay MAZ expression in high and low metastatic cell lines. 3. Building MAZ lentiviral vector transfected cells sune1-5-8F and sune1-6-10 B. Transwell, scratch assay were employed to estimate MAZ expression on nasopharyngeal carcinoma cell invasion and metastasis. 4. Co-immunoprecipitation(co-IP), Real-time PCR and Western Blot were used to detect the interactive relationship between MAZ and c-Myc expression and change of downstream related gene.Results1. 5-year Progression-free survival, overall survival, distant metastasis-free survival and local recurrence-free survival rates in patients with nasopharyngeal carcinoma were 56.8%, 68.2%, 68.7% and 92.8%. The total numbers of death were 74 cases,67 cases of cancer-related death, of which 14 cases(20.9%) died due to bleeding nasopharynx, 53 cases(79.1%) died of distant metastasis. Locoregional recurrence 26 cases(11.2%), distant metastasis 64 cases(27.5%). Distant metastasis is the main reason for the failure. Univariate and multivariate analysis showed that N stage was an independent risk factor for distant metastasis-free survival. 2. Immunohistochemical staining showed that MAZ expression in metastasis tissue were significantly higher than those without metastasis tissue(P <0.01), which was consistent with the microarray results; MAZ expression and clinical data analysis showed: in distantmetastasis nasopharyngeal carcinoma, MAZ increased expression was correlation with N staging(P <0.05). Real-time PCR and Western Blot showed MAZ was highly expressed in high metastatic cell lines. 3. Successfully constructed stable cell lines with lentiviral vector.MAZ knockout inhibited NPC metastasis and invasion. 4. Co-immunoprecipitation prompted MAZ possibly through interaction with C-myc to adjust the MMP9, TGF-β, and COX-2, which promoted NPC metastasis.Conclusion1. After IMRT treatment, 5-year local regional control rate and overall survival, were 92.8% and 68.2%, distant metastasis is the main reason of failure. Distant metastasis-free survival rate was related to N staging. In metastatic tissue of nasopharyngeal carcinoma and highly metastatic cells,the MAZ expression was significantly upregulated, closely related to NPC N staging. 2. MAZ expression promoting NPC cell metastasis and invasion. 3. MAZ maybe interact with the C-myc to adjust the MMP9, TGF-β, and COX-2, which promote NPC metastasis.