Functional Study Of TRIM24 And PLU-1 In Head And Neck Squamous Cell Carcinoma

【Aim】Head and neck squamous cell carcinoma(HNSCC) is one of the most common cancer types worldwide. Despite treatment advances in the past few decades, the clinical outcomes of patients with advanced HNSCC remain dismal. Therefore, further elucidating the molecular basis of HNSCC will be critical to identify molecular abnormalities contributing to HNSCC progression. These abnormalities may serve as biomarkers for predicting clinical outcomes and/or molecular targets for better treatment of patients with HNSCC. After HNSCC candidate gene selection, an E3 Ubiquitin ligase TRIM24 and a histone demethylase PLU-1 were found to be playing roles in the HNSCC progression. So the aim of this study was to investigate TRIM24 expression and its clinical significance in HNSCC and to investigate the role of PLU-1 in cell proliferation and cisplatin resistance in HNSCC cell lines and patients.【Method】 For the first part, The expression levels of TRIM24 variants were examined in HNSCC samples and cell lines by real-time PCR and Western Blot. The expression levels of TRIM24 were measured in 91 locally advanced HNSCC tumors by immunohistochemistry(IHC) and were correlated with clinical and pathological parameters. The functional role of TRIM24 in HNSCC was further investigated by silencing its expression in HNSCC cell lines. For the second part, our group previously predicted the interaction of NPM1 and PLU-1 protein with Snail protein. So immunoprecipitation and immunoflurescence assay were used to confirm the binding between the two noval proteins with Snail protein separately. Then expression levels of PLU-1 m RNA and protein were examined in HNSCC cell lines, oral leukoplakia tissues and HNSCC tissues using realtime-PCR and IHC. Chromatin immunoprecipitation(Ch IP) assay was used to examine the promoter regulation of PLU-1 transcriptional activity. Stable PLU-1 knockdown HN4 and HN13 cells were established and used as in vitro models to examine the prolierative and cisplatin resistant roles of PLU-1 in HNSCC cells. Nude mice xenograft model were used to observe tumor gowth affected by PLU-1 knockdown in vivo.【Results】 In the first part, TRIM24 variants were up-regulated in 56 HNSCC samples(p <.001) and 9 HNSCC cell lines(p < 0.05) compared to adjacent normal tissues and normal oral epithelial cells. TRIM24 protein was overexpressed in 6 of the 8 HNSCC cell lines and in 2 of the 3 HNSCC tissue samples examined. Furthermore, 54.95%(50/91) of HNSCC tissue samples exhibited remarkably elevated expression of TRIM24 by IHC. Univariate analysis revealed that the higher TRIM24 expression was associated with adversed worse overall survival of patients with HNSCC(p = 0.020). In multivariate analysis, TRIM24 expression was identified as an independent predictor of overall survival(p = 0.030), after adjusting for other clinicopathological parameters. Upon TRIM24 silencing, the growth of HNSCC cells was notably reduced due to the increased of apoptosis. In the second part, NPM1 mutant were confirmed to bind with Snail protein, which enhanced the repression of E-cadherin transcriptional activity. While PLU-1 were found not interacting with Snail protein or functioning in the EMT process. Interestingly, PLU-1 expressed at moderate to high levels in 8 HNSCC cell lines and 1 immortalize oral epithelial cell line, 26 oral leukoplakia tissues and 99(88% considered as high level or positive expression) HNSCC tissues, whereas only weak expression could be detected in normal oral epithelial cells and oral mucosa from healthy individuals. Transcription factor Sp1 could bind to the promoter of PLU-1 gene and negatively regulate its transcriptional activity. Futhermore, positive expression of PLU-1 is associated with higher levels of Ki-67 in 26 HNSCC samples examined(Pearson r = 0.6514, p = 0.0003). PLU-1 silenced HN4 and HN13 cells displayed a reduced cell growth rate both in vitro and in vivo. Knockdown of PLU-1 led to to G1 arrest and apoptosis induction in HNSCC cells, and enhanced cisplatin sensitivity in resistant HNSCC cell lines.【Conclusion】Aberrant TRIM24 expression may play an important role in the development of HNSCC and is a promising prognostic indicator for patients with locally advanced HNSCC. PLU-1 is overexpressed in HNSCC and the expression levels correlates with Ki-67 expression levels. PLU-1 may play a role in cell proliferation and apoptosis with a potential implication in modulating cisplatin resistance in HNSCC.