Abstract 3967: Identification of metadherin as a novel target of miR-375 in head and neck cancer

Abstract

Abstract Purpose: The objective of this study is to investigate the significance of miR-375 and its target metadherin in head and neck cancer (HNC) patients. Methods and Results: Expression of miR-375 was measured by quantitative RT-PCR and observed to be significantly under expressed in both head and neck squamous cell (HNSCC) and nasopharyngeal carcinoma (NPC) samples by factors of 5.0 and 4.4, respectively. One mechanism of miR-375 silencing appears to be promoter methylation. Using a combination of experimentally-observed transcript changes after miR-375 re-expression in HNC cell lines, with in silico prediction algorithms, metadherin was identified as a novel target of miR-375. Luciferase reporter assays confirmed an inhibitory interaction between miR-375 and the 3’UTR of metadherin. Functional characterizations of miR-375 and metadherin were then conducted using FaDu (hypopharyngeal squamous cell) and C666-1 (nasopharyngeal) cancer cell lines. In vitro re-expression of miR-375 as well as knock-down of metadherin using siRNA markedly decreased cell viability and clonogenic survival (30% – 45%), cell migration (50% – 80%), and in vivo tumour forming ability in both cancer models. In vitro cell invasion was also significantly decreased by miR-375 over-expression and metadherin knock-down in FaDu cells (>90%). Western blot analyses demonstrated that metadherin knock-down resulted in reduced phosphorylation of PKB, indicating that the PI(3)K pathway may be involved in mediating these effects. Using qRT-PCR on 100 NPC and 22 HNSCC archival samples, there was an inverse relationship between metadherin over-expression with miR-375 under-expression, corroborating the relevance of this axis in primary human HNC tissues. Furthermore, NPC patients whose tumours expressed high levels of metadherin (defined as > median) experienced significantly lower overall and disease-free survival (5-year OS of 82%, DFS of 73%) compared to patients with low (< median) expression levels (5-year OS of 66%, DFS of 56%). Specifically, this reduced survival was most significantly related to distant metastases, wherein high metadherin expression was significantly associated with distant relapse (5-year distant relapse rates: 26% vs. 5% for high vs. low expressors, respectively). Conclusions: We report that the miR-375:metadherin control mechanism may represent a novel oncogenic pathway driving human HNC progression, potentially mediated in part by the PI(3)K cascade. Most importantly, metadherin over-expression was a significant predictor of distant metastases, a major clinical challenge in HNC given the improvements in loco-regional control as a consequence of technical advances in radiation therapy delivery. Hence, targeting this pathway could be a potentially novel therapeutic strategy to improve outcome for HNC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3967. doi:10.1158/1538-7445.AM2011-3967