Oncogenic Roles Of Bmi1and Its Therapeutic Inhibition By Histone Deacetylase Inhibitor In Tongue Cancer

Background:The polycomb complex protein Bmi1(B lymphoma Mo-MLV insertion region 1 homolog) mediates epigenetic transcriptional silencing by modifying chromatin structure and has been found to be involved in diverse fundamental cellular processes, such as cell proliferation, apoptosis, senescence, epithelial–mesenchymal transition(EMT) and stem cell maintenance.Bmi1 is frequently overexpressed in human malignancies and therefore has key diagnostic and prognostic significance, and holds potential as a therapeutic target. In order to clarify the related biological effects of Bmi1 in TSCC, we designed and carried out the following experiments: Objectives:To characterize the expression patterns and oncogenic roles of Bmi1 in tongue squamous cell carcinoma and to determine the anticancer effects of histone deacetylase inhibitors(HDACi) via Bmi1 inhibition against tongue cancer. Methods:(1) Immunohistochemical staining was used to survey the expression of Bmi1 and ki67 in TSCC samples and normal tongue specimens. The correlation of the Bmi1 level with patients’ clinicopathological characteristics and overall survival was further assessed.(2) The cell phenotype was verified by Q-PCR and Western blot, MTT assay, and Transwell assays which were used to compare the role of OSCC cells in promoting tumor cells proliferation migration, apoptosis, senescence, colony forma tion, CD44+CD133+ sub-population, cisplatin chemosensitivity and EMT after Short-hairpin RNA-mediated Bmi1 knockdown.(3) TSCC cells were observed relate-d to biological characteristics by histone deacetylase inhibitor TSA and Na B treatment.(4) By a tongue cancer xenograft model, we observed the effect of Genetic Bmi1 silencing and pharmacological inhibition of Bmi1 by Na B treatment in tumor growth. Results: Bmi1 m RNA levels in the cancerous cells except the Tca8113 were significantly higher than that in normal mucosa as assessed by real-time RT-PCR assay. The amounts of Bmi1 m RNA in Cal27、SCC9、SCC25、HN4、HN6、HN12 were elevated approximately 3.45、3.71、3.57、8.42、5.82 and 6.21-folds(P<0.01), respectively. The following Western blotting results further confirmed the significantly upregulated Bmi1 expression in the TSCC cells lines except Tca8113. Bmi1 was readily detected and mainly identified in nucleus by cellular immunofluorescence. We next evaluated the expression patterns of Bmi1 by immunohistochemical staining in a retrospective cohort of 52 primary TSCC samples. The results indicated Bmi1 abundance in these TSCC specimens were graded as low(16) or high expression group(36), whereas its expression levels in normal counterparts were divided into negative(4), low(9) and high(3) group, a clear indicative of aberrant overexpression of Bmi1 in a major fraction of TSCC samples(P<0.01). There were no significant correlations between Bmi1 expression with patients’ age, gender, tumor size, local invasion, pathological grade and clinical stage. Noticeably, significant association between Bmi1 abundance was associated with cervical nodes metastasis(P=0.0367) and Ki-67 expression(P=0.0384). In a Kaplan-Meier survival analysis, high Bmi1 expression in TSCC was significantly associated with reduced overall survival(log-rank, P=0.0179), suggesting that Bmi1 overexpression associated with adverse prognosis.(2) Short-hairpin RNA-mediated Bmi1 knockdown inhibited cell proliferation and migration, induced cell apoptosis and senescence, reduced colony formation and CD44+CD133+ sub-population as well as enhanced cisplatin chemosensitivity, presumably by modulation of p16, p14 and E-cadherin.(3) HDACi chemicals Trichostatin A(TSA) and sodium butyrate(Na B) potently inhibited Bmi1 and triggered similar phenotypic changes reminiscent of Bmi1 silencing, although TSA treatment seemed paradoxically to induce some epithelial–mesenchymal transition-like changes in tongue cancer cells. Importantly, Na B-induced antitumor effects were partially attenuated by enforced Bmi1 overexpression in vitro.(4) Genetic Bmi1 silencing and pharmacological inhibition of Bmi1 by Na B treatment significantly impaired tumor growth in a tongue cancer xenograft model. Conclusion:Bmi1 is aberrantly overexpressed in human TSCC specimens and TSCC cell lines. Their overexpression is found to be significantly associated with cervical lymph node metastasis, and overall survival, suggesting that Bmi1 serves as a key driver with multiple oncogenic functions during tongue cancer progression and as a novel biomarker for diagnosis and prognostic prediction for patients. HDACi chemicals such as Na B induce therapeutic effects against tongue cancer probably in part by Bmi1 repression.