MiR-34c Downregulation Leads to SOX4 Overexpression and Cisplatin Resistance in Nasopharyngeal Carcinoma

Abstract

Background: A major cause of disease-related death in nasopharyngeal carcinoma (NPC) is the development of distant metastasis (DM) despite combination chemo-radiotherapy. We have previously identified and validated a four micro-RNA (miRNA) signature which was prognostic for DM.In this study, characterization of a key component of this signature, miR-34c, revealed its role in chemotherapy resistance. Methods: 246 NPC patient biopsies samples were subject to comprehensive miRNA profiling and immunohistochemistry (IHC). Two human normal nasopharyngeal cell lines (immortalized; NP69 and NP460), as well as the NPC cell line C666-1, were used to generate pre-miR-34c (gain of function) and anti-miR-34c (loss of function) cells. Signaling pathways were assessed using quantitative real-time PCR (qRT-PCR) and Western blot. Cell viability was measured using the ATP lite assay. Results: MiR-34c was downregulated in NPC patient samples, and confirmed in vitro to directly target SOX4, a master regulator of epithelial-to-mesenchymal transition (EMT). MiR-34c downregulation triggered EMT-representative changes in NP69 and NP460; Snail, ZEB1, CDH2, and SOX2 were upregulated, while Claudin-1 and CDH1 were downregulated. Phenotypically, inhibition of miR-34c led to cisplatin resistance, whereas miR-34c over-expression sensitized NPC cells to cisplatin.TGFβ1 decreased miR-34c and increased SOX4 expression in vitro. The TGFβ receptor 1 inhibitor SB431542 reduced SOX4 expression and increased cisplatin sensitivity. Finally, immunohistochemistry revealed that in chemotherapy-treated NPC patients, lower SOX4 expression was associated with improved overall survival. Conclusion: Taken together, miR-34c downregulation correlates with higher incidence of DM; miR-34c also increases SOX4 expression which leads to cisplatin resistance. In association, TGFβ1 represses miR-34c expression, and inhibition of the TGFβ1 pathway is one strategy to restore cisplatin sensitivity in NPC. BackgroundNasopharyngeal carcinoma (NPC) patients presenting with locally advanced disease have a very modest overall survival (OS) rate: approximately 65% after 5 years [1][2][3]. Despite the use of intensitymodulated radiation therapy for this Epstein-Barr virus (EBV)-associated malignancy, 20-30% of NPC patients will still succumb to distant metastasis (DM) [4]. Therapeutic options for such NPC patients 4 are limited, and a primary clinical challenge is resistance to chemo-radiation [5].Concurrent chemotherapy (cisplatin/5-fluorouracil) with radiation therapy (RT) modestly improve OS, but can cause significant toxicity and death [4,[6][7][8][9][10].Our group previously completed a global miRNA NPC patient sample profiling, discovering and validating a four-miRNA prognostic signature for DM (low miR-34c, low miR-140, high miR-154, and high miR-449b) [11]. A subsequent study demonstrated that elevated levels of miR-449b was significantly associated with poor OS in patients receiving concurrent chemo-radiotherapy [12]. MiR-449b overexpression in NPC was found to decreas...