Expression Of RUNX3, Methylation Status And The Association With Clinicopathological Parameters And Prognosis In Patients With Salivary Gland Adenoid Cystic Carcinomas

Part one: Effect of 5-Aza-dc on RUNX3 expression in salivary gland adenoidcystic carcinoma cell lines ACC-2, ACC-3 and ACC-MAdenoid cystic carcinomas (ACC)are one of the most common malignancies insalivary gland, and comprise approximately 10% of all epithelial salivary neoplasms. Itis characterized by slow growth, followed by high neural and blood vessal invasion. Thedistant metastasis rate is appropriately 40% which results in a poor long term survival.Surgery followed by postoperative radiation therapy is the treatment of choice. It isreported that alternation of the antioncogen and cell adhesion molecules has beenproposed as factors associated with the aggressive behavior in salivary gland adenoidcystic carcinoma. However the underlying mechanism for the aggressive nature and thefatal course of this tumor are pooly investigated. The human runt-related transcriptionfactor-3 (RUNX3) is a tumor suppressor gene in gastric cancer. The inhibition orsilence of this gene expression is supposed to be highly associated with invasion anddistant metastasis of a variety of malignant tumor cell lines, whilst, the promotermethylation of RUNX3 gene is the main cause. However, after using the DNAmethyltransferase enzyme inhibitor, 5-Aza-dc, the expression of this gene can berestored in vitro. Promoter methylation is a common mechanism to inactivate tumorsuppressor genes in tumorigenesis, which plays an important role in the development ofsalivary gland tumors; however, the expression of RUNX3 in salivary tumor cell lines israre. Thus, it is necessary to investigate the expression of RUNX3 gene and protein insalivary gland adnoid cystic carcinoma cell lines, namely ACC-2, ACC-3, and ACC-M.Experiment one: in this part, RT-PCR, laser scanning confocal microscope (LSCM),and western blot were used to detect the expression of RUNX3 gene and protein insalivary gland adenoic cystic carcinoma cell lines, ACC-2, ACC-3, and ACC-M,before/after treated with 5-Aza-dc, restropectively.Three adenoid cystic carcinoma cell lines (ACC-2, ACC-3, and ACC-M) were usedin this vitro experiment. Those cell lines were cultured in RPMI-1640 with 10% fetalbovine serum, and incubated them at 37℃in a humidified atmosphere containing5%CO₂. 5-Aza-dc (300nmol/L) was added into the medium, and the cell lines were cultured together with 5-Aza-dc for 72 hours. After that the total RNA and proteinswere isolated from the cultured cells with or without treated with 5-Aza-dc. RT-PCRand western blot were used to detect the RUNX3 mRNA and protein expression in thesethree cells lines, respectively. In western blot analysis, the relative level of RUNX3protein was decided by the ratio between RUNX3 gradation and GAPDH gradationwith using Image-Pro Plus software.Results: RT-PCR and western blot analysis showed weak expression of RUNX3mRNA and protein in ACC-2 and ACC-3, and no expression of RUNX3 in ACC-3 cellline. The western blot analysis disclosed a RUNX3 protein of 45kDa in the three ACCcell lines. After treated by 300 nmol/L 5-Aza-dc for 72 hours, the expression level ofRUNX3 in ACC-2 and ACC-3 was improved, and in ACC-M was restored by analyzingthe gradation ratio of RUNX3 protein and GAPDH protein. A similar result was foundbetween RT-PCR and western blot analysis. The results of LSCM showed that theRUNX3 protein located mainly in the cytoplasm of the ACC cell lines. After treatedwith 300nmol/L 5-Aza-dc for 72 hours, both nuclear and cytoplasmic location ofRUNX3 positive signals were found in the ACC-2 and ACC-3 cells. However, the weakpositive signal was still only found in the cytoplasm of ACC-M cellsIn this part, the expression of RUNX3 gene and protein in salivary gland ACC celllines, ACC-2, ACC-3, and ACC-M, before/after treated with 5-Aza-dc, was exploredrestropectively. These results indicate that the promoter methylation of RUNX3 mightplay an important role in the inhibition of RUNX3 expression in ACC cell lines. Thesilence expression of RUNX3 in high lung metastasis salivary adenoid cystic carcinomacell line, ACC-M, provide a clew that the RUNX3 inhibition is associated with distantmetastasis of ACC. Because RUNX3 can fuction as a tumor suppressor when locates inthe nuclear of cells, the cytoplasmic mislocalization of RUNX3 in ACC cells might becorrelated with the development and progression of ACC.Part two: Promoter methylation and protein expression of the RUNX3 gene inthe clinicopathologic assessment of salivary gland adenoid cystic carcinomaThe methylation of CpG island in promoter region is the main mechanism of theRUNX3 gene silence. In part one, the results showed that the RUNX3 expression waselevated in adenoid cystic carcinoma cell lines ACC-2, ACC-3, and ACC-M after being treated by 5-Aza-dc, which indicates the association between promoter methylation inRUNX3 gene and the progression of salivary gland adnoid cystic carcinoma. As a tumorsuppressor, the RUNX3 expression was inhibited due to the promoter methylation in avariety of malignant tumors. However the mechanism of inhibition or sitence ofRUNX3 gene in salivary gland adenoid cystic carcinoma is pooly investigated. Thus, inthis chapter, the methylation status in successive ten regions ranging from the 5' regionto the transcription start site within the RUNX3 CpG island and the expression levels ofRUNX3 protein in ACC samples and compared non-neoplastic salivary glands wereexplored, and the relationship between the methylation status of RUNX3 gene andRUNX3 protein expression or the clinicopathological parameters was also analyzed, aswell as the mechanism of the critical regions and spreading of the RUNX3 CpG islandmethylation in ACC.Experiment two: The quantitive MSP method was used to detect the methylationstatus of CpG island in various regions (No. 1-10) of RUNX3 promoter region, and theWestern blot method was used for detecting the expression of RUNX3 protein in 41salivary gland ACC samples and the corresponding non-neoplastic salivary glands. ALogistic ANOVA model is used to analysis the risk ratio between the methylation statusof CpG island in RUNX3 gene and development of salivary ACC, meanwhile, thepossible association among the methylation of RUNX3 gene, the clinicopathologicalparameters of ACCs, and RUNX3 protein expression was compared.Results: The results of qMSP show that the hypermethylation initially occurs atthe most 5' region of the RUNX3 CpG island and spreads to the transcription start site.And the methylation rate is highest in region No. 1 and No. 2 among the successive tenregions ranging from the 5' region to the transcription start site within the RUNX3 CpGisland, and lowest in the transcription start site both in ACCs and normal salivary glands.Furthermore, there is no methylation in the transcription start site in nomal salivaryglands tissues. Together with the results of Logistic ANOVA model analysis, thoseresult indicats that the transcription start site within the RUNX3 promoter CpG island iscritical for gene silencing. Western blot results reveal that the RUNX3 protein level inACC was significantly lower than in normal salivary glands(P<0.001), in combinationthe results of qMSP, it is presumed that the RUNX3 gene methylation is one of the reason inducing the down-regulation of RUNX3 in ACCs. Although there is norelationship between the promoter methylation of RUNX3 CpG island and theclinicopahtological parameters of ACCs, analysis the methylation status at thetranscription start site of the RUNX3 CpG island can be used in diagnosis and riskassessment of ACCs.The promoter methylation is a common mechanism in the inactivation of tumorsuppressor genes in varieties of malignancies. In this study, we found the rate ofpromoter methylation of RUNX3 CpG island in salivary gland ACCs is significantlyhigher than it is in normal salivary gland, suggesting that the promoter methylation ofRUNX3 gene may be associated with the development of salivary gland ACCs. TheqMSP results reveal that the methylation ratio of RUNX3 CpG island differs in differentregion, hypermethylation initially occurs at the most 5' region of the RUNX3 CpGisland and spreads to the transcription site, which is supposed to be the critical site forgene silencing. Thus, the region spanning the transcription site, such as No. 6-8, shouldbe used to evaluate the methylation status of RUNX3 gene in tumors. Additionally, Norelationship between hypermethylation of RUNX3 gene and clinicopathologicalparameters in patients with salivary gland ACCs was found. A large clinical trial wasrequired to further evaluate the potential relationship.Part three: Expression of RUNX3 in salivary gland adenoid cystic carcinomaand its association with tumor progress and prognosisThe long-term survival in patients with salivary gland adenoid cystic carcinoma isgloomy. Various parameters, including positive surgical margins, clinical stage,histopathological patterns, and perineural invasion, have been reported as relevantprognostic factor in patients with ACC. In addition to these chinicopathological factors,several investigators have explored the possible association between the proliferation orapoptosis-associated proteins and the survival in patients with salivary ACC. However,the precise mechanism responsible for its carcinogenesis has not been fully clarified.RUNX3 gene, supposed to be a new tumor suppressor, the silence expression of thisgene is highly correlated with the development and progress of gastric cancer, as well asthe therapy outcome and the survival in patients with gastric carcinomas. To detect theexpression of RUNX3 in a variety of malignant rumors might provide a better prognostic indicator. Moreover, the lack of RUNX3 expression is probably associatedwith distant metastasis in malignancies, such as gastric cancer. Although there is noreport on its expression in salivary ACC, which presents typically distant meatstasisresulting in a fatal outcome, on basis of the relationship between the RUNX3 expressionand distant meatastasis, RUNX3 expression might be conceivably associated with thetumorigenesis, progression, distant meatastasis, and survival of salivary ACC.Experiment three: The quantitative RT-PCR (qRT-PCR), Western blot andimmunohistochemistry were applied for detecting the expression of RUNX3 in normalhuman salivary glands and adenoid cystic carcinomas in this part.Nine normal adult salivary glands (including 3 parotid glands, 2 sublingual glands,and 2 submandibular glands) and seven adenoid cystic carcinomas frozen tissues wereobtained. The expressions of RUNX3 mRNA and protein were detected by usingqRT-PCR and western blot analysis, respectively. The relative RUNX3 mRNA levelwas measured by using the threshold cycle (Ct) method. In western blot analysis, therelative level of RUNX3 protein was decided by the ratio of RUNX3 gradation andGAPDH gradation with using Image-Pro Plus software. Seventy-three formalin-fixed,paraffin-embedded salivary ACC specimens were achieved. The immunohistochemistrywas applied to detect the subcellular location of RUNX3 protein in normal salivaryglands and ACC.Results: The relative quantification of RUNX3 mRNA expression in ACC(0.09±0.05) was two-fold lower than that in normal salivary glands (0.19±0.08).Western blot analysis disclosed a RUNX3 protein of 45 kDa both in human normalsalivary glands and ACC.According to the results of gradation analysis, the relative ofRUNX3 protein expression in salivary gland ACC (0.79±0.03) was lower than that innormal salivary glands (0.88±0.03). However, no statistical difference between thesetwo groups. Immunohistochemistry results showed that RUNX3 protein located mainlyin the nuclear and cytoplasm of acinous cell and ductal cell in normal salivary gland;and only in the cytoplasm of tumor cell in ACC. In some cases of solid type ACC, noexpression RUNX3 protein was found.Experiment four: The potential relationship between the expression of RUNX3 andthe clinicopathological factors and survival in patients with adenoid cystic carcinoma was analyzed in this part.The semi-quantative RUNX3 level in 73 adenoid cystic carcinoma cases was detectedwith using Image-Pro Plus software. Continuous variables of RUNX3 labeling indexwere divided into three discrete exclusive variables according to the distribution of thesamples. The tertile cut-off values were as follows: High group (≥14.91%), intermediategroup (6.94%-14.91%), and low group (≤6.94%). Significant differences between theexpression of RUNX3 and clinicopathological parameters were compared. Theunivariate analysis with the Cox regression model was used to determine identifiedprognostic factors, and multivariate analysis with the Cox regression model was used toexplore combined effects. Survival analysis was computered by means of theKaplan-Meier method and significant levels were assessed by means of the log-rank test.Thereby, the application of RUNX3 expression in salivary gland ACC to predict thepatients' survival was assessed.Results: The semi-quantitative results of immunohistochemistry showed that the high,intermediate, and low RUNX3 expressions were 19.60±4.26%, 11.19±2.42%, and4.78±1.59%, respectively. And the RUNX3 expressions were 13.69±6.46% in thecribriform pattern, 11.78±4.40% in the tubular pattern, and 8.86±5.20% in the solidpattern. Low RUNX3 expression was significantly correlated with the histologicalpatterns of ACC, namely solid subtype (P=0.025), meanwhile, ACC with lower RUNX3expression significantly tended to be have more frequent distant metastasis (P=0.009),indicating the the association between down-regulation of RUNX3 expression andtumor progression. The RUNX3 expression percentage was 13.65±6.44% in tumorswith stage T1-3, and 10.24±5.19% in stage T4. There is only a weak tendency betweenthe low RUNX3 expression and stage T4 (P=0.061). No statistical differences betweenRUNX3 expression and other clinicopathologic factors, such as age, gender, tumor site,perineural invasion, and lymph nod involvement in ACCs, was detected. The 5-yearsurvival rate was 90% in the 18 patients with high levels of RUNX3 expression, 75.4%in the 37 patients with moderate level, and 48.27% in the 18 patients with lowlevel.Using univariate analysis of Cox regression model, the following variables werefound to be significantly associated with a worse prognosis, including low expression ofRUNX3 protein (P=0.012), lymph node involvement (P=0.007), and distant metastasis (P<0.001). Kaplan-Meier survival curves showed that the survival rates of patients withlow expression of RUNX3 was significantly worse than that of patients with moderateor high RUNX3 expression (P=0.022). Meanwhile, we observed that some otherclinicopathogical parameters, including stage T4 (P=0.003), lymph node involvement(P=0.004), and distant metastasis (P<0.001) were significantly related with decreasedsurvival. However, no significant association between overall survival and solidhistotype (P=0.771) or perineural invasion (P=0.554) was found. Multivariate survivalanalysis revealed that only distant metastasis was an independent significant prognosticfactor (P=0.043). Stage T4 only had a weak association with overall survival (P=0.063).RUNX3 gene, as a tumpr suppressor, is signigicantly associated with the survival rateand prognosis in patients with malignancies, and might be an independent predictor forthe prognosis. The 5-year survival rate is significant higher in tumor with highexpression of RUNX3 protein than this with low expression level. Meanwhile, theexpression of RUNX3 protein is correlated with cell differention, furthermore, the lowor lack expression of RUNX3 is potentially associated with distant metastasis. Theresults of this study indicate that the low expression of RUNX3 in salivary gland Aceis correlated with tumor development and metastasis. Together, the expression ofRUNX3 protein might be a candidate for diagnosis and prognostic marker in salivarygland ACCs. It has a pivotal role in the tumor proliferation and apoptosis.Conclusions:1. There is RUNX3 expression in adenoid cystic carcinoma cell lines ACC-2 andACC-3, and silence of RUNX3 in ACC-M. The DNA methyltransferase enzymeinhibitor, 5-Aza-dc, can elevate the RUNX3 level among these three cell lines.2. The incidence of promoter methylation in RUNX3 gene CpG island in humansalivary gland adenoid cystic carcinoma is significanctly higher that it is innon-neoplastic salivary gland, indicating that the promoter methylation of RUNX3gene plays an critical role in the development of this malignancy.3. The promoter methylation of RUNX3 CpG island spreads frorn the most 5' regionto the rranscriptiong start site in human salivary gland ACC, the incidence rate ofpromoter methylation in the transcriptiong start site is the lowest, suggesting this site might be a critical region for the methylation of RUNX3 gene.4. There is no significant difference between the methylation status of RUNX3 geneand the clinicopathological parameters in patients with ACCs.5. The expression of RUNX3 protein is down-regulated in salivary gland ACC. Thelower expression of this protein is significantly associated with rumor differentiation,distant metastasis, and prognosis. The expression of RUNX3 protein has a definitevalue in judging prognosis in salivary gland ACC.