Abstract 4932: Role of ribonucleotide reductase subunit M2 in regulation of head and neck and lung cancer undergoing invasion.

Abstract

Abstract Ribonucleotide reductase subunit M2 (RRM2) is a key enzyme in DNA replication which can serve as a potential therapeutic target. RRM2 is also thought to modulate cellular invasiveness but the mechanisms through which RRM2 affects the invasive phenotype have not been elucidated. In this study, we report a mechanism linking RRM2 to invasion which provides a vital clue for the development of novel therapeutics. Since RRM2 is a known prognostic factor and an indicator of poor patient outcome in several cancers, we investigated the correlation between RRM2 and metastasis in tumor tissues from head and neck squamous cell carcinoma (HNSCC) patients. We utilized both quantum dot (QD)-immunofluorescence and conventional immunohistochemistry techniques to stain RRM2 proteins in metastatic (n = 20) and non-metastatic (n = 20) HNSCC tissue samples. In addition, we validated the clinical findings in an in vitro system and explored the underlying mechanism in invasion. The function of RRM2 in cell invasion was examined in different HNSCC cell lines (Tu212, Tu686, 37A, M4e, 886LN) and non-small cell lung cancer (NSCLC) cell lines (A549, H460 and H1299). siRNA was applied to knockdown the RRM2 protein while pcDNA3/RRM2 plasmid was used to establish RRM2 overexpressing cell lines. Cell invasion was examined by Boyden chamber invasion assay. Molecular signaling analysis was performed by Western blotting. Our results showed that RRM2 had a significant positive correlation with metastasis in HNSCC tumor tissues (p value < 0.01). We also identified varying degrees of RRM2 expression levels in HNSCC and NSCLC cell lines, which were correlated with their invasive capability. Highly invasive HNSCC (M4e) and NSCLC (H1299) cell lines that feature epithelial-mesenchymal transition (EMT) expressed the highest level of RRM2 protein and exhibited the greatest invasive ability among the cell lines studied. Upon knockdown of RRM2 by siRNA treatment, we found more than 3-fold reduction of invasive ability of M4e and H1299 cells compared to non-targeted siRNA control (p value < 0.001). In contrast, overexpression of RRM2 enhanced cell invasiveness 2 to 4-fold in poorly invasive HNSCC (Tu212 and Tu686) and NSCLC (A549) cell lines compared to empty vector (p value < 0.001). We determined that RRM2 regulated EMT features and identified molecules, such as E-cadherin, vimentin, Twist1, caveolin-1, Akt, and Erk, which are associated with this biological process. Our study demonstrated that RRM2 substantially promoted cell invasion. Thus, an elevated RRM2 may serve as a potential biomarker for metastasis. Targeting RRM2 and its downstream signaling intermediaries represents a rational approach for developing novel anticancer therapeutics. (This work is supported by grants from NIH U01CA151802, P50CA128613 and DOD W81XWH-07-1-0306) Citation Format: Mohammad Aminur Rahman, A.R.M. R. Amin, Sreenivas Nannapaneni, Zhongliang Hu, Hyunseok Kang, Zhuo (Georgia) Chen, Dong M. Shin. Role of ribonucleotide reductase subunit M2 in regulation of head and neck and lung cancer undergoing invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4932. doi:10.1158/1538-7445.AM2013-4932