Abstract 1428: Targeting RRM2 by siRNA inhibits cellular invasion and represents a rational approach for inhibition of metastasis of head and neck and lung cancers
Authors: Rahman Mohammad Aminur , Amin A.R.M. Ruhul , Zhang Jun , Nannapaneni Sreenivas , Saba Nabil F. , Chen Zhuo Georgia , Shin Dong M. 
Published: August 2015

Background: Ribonucleotide reductase subunit M2 (RRM2) has been frequently observed to be aberrantly overexpressed in various tumors. RRM2 is a key enzyme, and essential regulator of balanced deoxyribonucleotides (dNTPs). It is critical for DNA replication and repair, and hence cell survival. In addition to DNA synthesis, it has been reported to modulate cellular invasiveness. However, the mechanisms through which RRM2 affects the invasive phenotype have not been elucidated.

Methods: We evaluated the expression of RRM2 protein in metastatic (n = 40) and non-metastatic (n = 40) head and neck squamous cell carcinoma (HNSCC) tissue samples using immunohistochemistry (IHC). We identified varying degrees of RRM2 expression levels in HNSCC and NSCLC cell lines. We also generated RRM2 overexpressing stable cell lines; Tu686 (HNSCC) and A549 non-small cell lung carcinoma (NSCLC). In addition, we knocked down RRM2 and Twist 1 expression by siRNA technology.

Results: Our results showed that RRM2 expression was significantly correlated with lymph node metastasis in HNSCC (p value &lt; 0.01). We also observed that 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, we observed more than 3-fold reduction of invasive ability of M4e and H1299 cells compared to non-targeted siRNA control (p value &lt; 0.001). In contrast, overexpression of RRM2 enhanced cell invasiveness 2 to 4-fold in poorly invasive HNSCC (Tu686) and NSCLC (A549) cell lines compared to empty vector (p value &lt; 0.001). We observed molecular modifications by utilizing Western blotting. We found that RRM2 overexpression significantly suppressed an epithelial marker E-Cadherin. Overexpression of RRM2 induced Twist 1 protein that regulates E-Cadherin and drives cellular migration and invasion. Our further mechanistic studies reveal that RRM2 plays role in Twist 1 induction by protein stabilization. We silenced Twist 1 gene to evaluate the biological effect and found that upon knocking down Twist 1, cellular invasion and migration was suppressed like silencing of RRM2.

Conclusions: Our study demonstrated that RRM2 substantially promoted cell invasion by stabilizing Twist 1 and suppressing E-Cadherin. Thus, an elevated RRM2 may serve as a potential biomarker for metastasis of HNSCC and NSCLC. Targeting RRM2 and its downstream signaling intermediaries may represent a rational approach for developing novel anticancer therapeutics. We are in the process of conducting an animal study to validate the strategy of silencing of RRM2 by using RRM2-siRNA nanoparticle to inhibit metastasis. (This work is supported by grants from NIH U01CA151802, P50CA128613).

Citation Format: Mohammad Aminur Rahman, A.R.M. Ruhul Amin, Jun Zhang, Sreenivas Nannapaneni, Nabil F. Saba, Zhuo Georgia Chen, Dong M. Shin. Targeting RRM2 by siRNA inhibits cellular invasion and represents a rational approach for inhibition of metastasis of head and neck and lung cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1428. doi:10.1158/1538-7445.AM2015-1428